Figure 7Correlation between gene expression and Alu RNA processing ratio for AD upregulated genes ( Dataset EV3 ) ( upper panel ) and a random set of non - differentially expressed genes ( Dataset EV5 ) ( lower panel ) . For every gene a correlation coefficient was calculated ( Pearson correlation ) together with the respective p - value . Statistical test is based on Pearson ' s product moment correlation coefficient and follows a t distribution using the cor . test function in R package stats . Based on the r and p value genes were classified into four categories : i ) NS , non - significant for p value > 0 . 05 , ii ) no correlation or weak correlation ( r ≤ 0 . 05 ) , iii ) significant ( p < 0 . 05 ) and strong correlation ( 0 . 5Expression levels of full length Alu RNA ( RT - qPCR ) in the Alu RNA KD experiment . Statistical significance ( p value threshold 0 . 05 ) for the comparison between anti - Alu LNA treated samples ( anti - Alu ) and samples treated with a scramble control LNA ( ctrl ) with p = 0 . 049 and n = 3 , unpaired non - directional t - test . Error bars represent standard deviation from the mean . Expression levels of P53 ( RT - qPCR ) in the Alu RNA KD experiment . Statistical significance ( p value threshold 0 . 05 ) for levels in anti - Alu LNA treated samples ( anti - Alu ) greater than samples treated with a scramble control LNA ( ctrl ) with p = 0 . 046 and n = 3 , unpaired directional t - test . Error bars represent standard deviation from the mean . Expression levels ( RT - qPCR ) _ of selected genes from panel A that are either strongly correlated with Alu RNA processing ( left , r > 0 . 5 ) or only weakly / not correlated ( right , r < 0 . 5 ) . Statistical significance ( p value threshold 0 . 05 ) for anti - Alu greater than control ( depicted as asterisk , n = 3 / group , unpaired directional t - test , error bars represent standard deviation from the mean ) . Pearson correlation co - efficient for each gene is depicted below the name of each gene .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7Correlation between gene expression and Alu RNA processing ratio for AD upregulated genes (Dataset EV3) (upper panel) and a random set of non-differentially expressed genes (Dataset EV5) (lower panel). For every gene a correlation coefficient was calculated (Pearson correlation) together with the respective p-value. Statistical test is based on Pearson's product moment correlation coefficient and follows a t distribution using the cor.test function in R package stats. Based on the r and p value genes were classified into four categories: i)NS, non-significant for p value > 0.05, ii) no correlation or weak correlation (r≤0.05), iii) significant (p < 0.05) and strong correlation (0.5Expression levels of full length Alu RNA (RT-qPCR) in the Alu RNA KD experiment. Statistical significance (p value threshold 0.05) for the comparison between anti-Alu LNA treated samples (anti-Alu) and samples treated with a scramble control LNA (ctrl) with p=0.049 and n=3, unpaired non-directional t-test. Error bars represent standard deviation from the mean. Expression levels of P53 (RT-qPCR) in the Alu RNA KD experiment. Statistical significance (p value threshold 0.05) for levels in anti-Alu LNA treated samples (anti-Alu) greater than samples treated with a scramble control LNA (ctrl) with p=0.046 and n=3, unpaired directional t-test. Error bars represent standard deviation from the mean.Expression levels (RT-qPCR)_of selected genes from panel A that are either strongly correlated with Alu RNA processing (left, r> 0.5) or only weakly/not correlated (right, r <0.5). Statistical significance (p value threshold 0.05) for anti-Alu greater than control (depicted as asterisk, n=3/group, unpaired directional t-test, error bars represent standard deviation from the mean). Pearson correlation co-efficient for each gene is depicted below the name of each gene.


Figure 8In vitro incubation of one of the Alu RNA consensus sequences for different incubation periods . In vitro transcribed Alu RNA ( 67nM ) incubated at 37oC with 250nM HSF1 in the course of 6 hours with time intervals of 1 hour . Comparison among Hsf1 ( ~ 60KDa ) , denatured HSF1 , Poly A polymerase ( ~ 56KDa ) and no protein ( just TAP buffer ) with regard to Alu RNA processing ( estimating relative full length RNA remaining ) ( two replicates ) . The full gels are available as Source Data For Figure 8 . Relative full length RNA remaining was calculated using ImageJ area under the curve software over time . Error bars represent standard deviation from the mean . Comparison among Alu RNA ( three replicates ) , and two control RNAs ( two replicates ) regarding the full - length RNA levels remaining after in vitro incubation for 90 minutes at 37C with HSF1 . Sizes of control RNAs are control for RNA # 1 , 143nt and for control RNA # 2 , 432nt . Incubation in the absence of HSF1 but presence of the same buffer ( TAP ) was used as control to take into account any non HSF1 specific RNA destabilization due to non - specific degradation . Threshold for statistical significance was a p value of 0 . 05 with p = 0 . 03 ( unpaired , non - directional t - test ) for the comparison between HSF1 and no protein incubation ( n = 3 , for Alu RNA ) . The full gels are available as Source Data For Figure 8 . Error bars represent standard deviation from the mean . Plotting of the position of the first base ( 5 ' end ) of Alu RNA fragments across the Alu consensus sequence produced by Alu RNA that has been processed in vitro for 90 minutes at 37C in the presence of HSF1 ( upper panel ) and compared with one of the in vivo samples of Figure 1 ( lower panel ) . The x axis represents an Alu RNA metagene aligned at the start site of the Alu consensus sequence and the y axis shows the 5 ' end count for Alu RNA fragments aligning to any position downstream of position + 1 . The in vivo sample x axis depicts a 11nt sift compared to the in vitro one . Scatterplot depicting the positive correlation between HSF1 mRNA expression values and Alu RNA processing ratio in MAP AD patients ( r = 0 . 72 , p < 0 . 001 ) . Statistical test is based on Pearson ' s product moment correlation coefficient and follows a t distribution using the cor . test function in R package stats . Scatterplot depicting lack of correlation between HSF1 mRNA expression values and full length Alu RNA levels in MAP AD patients ( ns = non - significant , with p value 0 . 05 as the significance threshold ) . Statistical test as in subfigure 8E .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 8In vitro incubation of one of the Alu RNA consensus sequences for different incubation periods. In vitro transcribed Alu RNA (67nM) incubated at 37oC with 250nM HSF1 in the course of 6 hours with time intervals of 1 hour.Comparison among Hsf1 (~60KDa), denatured HSF1, Poly A polymerase (~56KDa) and no protein (just TAP buffer) with regard to Alu RNA processing (estimating relative full length RNA remaining) (two replicates). The full gels are available as Source Data For Figure 8. Relative full length RNA remaining was calculated using ImageJ area under the curve software over time. Error bars represent standard deviation from the mean.Comparison among Alu RNA (three replicates), and two control RNAs (two replicates) regarding the full-length RNA levels remaining after in vitro incubation for 90 minutes at 37C with HSF1. Sizes of control RNAs are control for RNA #1, 143nt and for control RNA #2, 432nt. Incubation in the absence of HSF1 but presence of the same buffer (TAP) was used as control to take into account any non HSF1 specific RNA destabilization due to non-specific degradation. Threshold for statistical significance was a p value of 0.05 with p=0.03 (unpaired, non-directional t-test) for the comparison between HSF1 and no protein incubation (n=3, for Alu RNA). The full gels are available as Source Data For Figure 8. Error bars represent standard deviation from the mean.Plotting of the position of the first base (5' end) of Alu RNA fragments across the Alu consensus sequence produced by Alu RNA that has been processed in vitro for 90 minutes at 37C in the presence of HSF1 (upper panel) and compared with one of the in vivo samples of Figure 1 (lower panel). The x axis represents an Alu RNA metagene aligned at the start site of the Alu consensus sequence and the y axis shows the 5' end count for Alu RNA fragments aligning to any position downstream of position +1. The in vivo sample x axis depicts a 11nt sift compared to the in vitro one.Scatterplot depicting the positive correlation between HSF1 mRNA expression values and Alu RNA processing ratio in MAP AD patients (r=0.72, p<0.001). Statistical test is based on Pearson's product moment correlation coefficient and follows a t distribution using the cor.test function in R package stats. Scatterplot depicting lack of correlation between HSF1 mRNA expression values and full length Alu RNA levels in MAP AD patients (ns=non-significant, with p value 0.05 as the significance threshold). Statistical test as in subfigure 8E.


Figure 1A qPCR analysis of relative SCTR levels in different tissues [ heart ( He ) , intestine ( Int ) , inguinal WAT ( Ing ) , epididymal WAT ( Ep ) , brown fat adipose ( BAT ) , liver ( Liv ) , hypothalamus ( Hypo ) , placenta ( Pla ) , skeletal muscle ( Mus ) , kidney ( Kid ) ] from C57BL / 6J mice as indicated ( n = 5 per group ) . Data information : Statistical significance was determined using Two - tailed Student ' s t test . Data are presented as mean ± SD . * * p < 0 . 01 ; * * * p < 0 . 001 . B Correlation of serum SCT levels and serum triglycerides ( TG ) , total cholesterol ( TC ) and nonesterified fatty acids ( NEFA ) levels in pregnant women in the middle trimester to the early third trimester ( n = 15 ) . Data information : Statistical significance was determined using Two - tailed Student ' s t test . Data are presented as mean ± SD . * * p < 0 . 01 ; * * * p < 0 . 001 . C , D Plasma SCT levels of dams ( C ) at gestational day 18 . 5 ( GD18 . 5 ) and embryo ( D ) at E18 . 5 ( n = 8 per group ) . Data information : Statistical significance was determined using Two - tailed Student ' s t test . Data are presented as mean ± SD . * * p < 0 . 01 ; * * * p < 0 . 001 . E Representative images of SCT immunohistochemical staining of duodenum from dams on standard ( CON ) , whole grain ( WG ) and high fat ( HFD ) diet at GD18 . 5 . SCT expression of WG is highest , but lower in HFD compared with CON . Scale bar , 50 μm ( n = 10 per group ) . Right part shows quantification of SCT positive cells in duodenum from dams of three groups . Data information : Statistical significance was determined using Two - tailed Student ' s t test . Data are presented as mean ± SD . * * p < 0 . 01 ; * * * p < 0 . 001 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A qPCR analysis of relative SCTR levels in different tissues [heart (He), intestine (Int), inguinal WAT (Ing), epididymal WAT (Ep), brown fat adipose (BAT), liver (Liv), hypothalamus (Hypo), placenta (Pla), skeletal muscle (Mus), kidney (Kid)] from C57BL/6J mice as indicated (n = 5 per group). Data information: Statistical significance was determined using Two-tailed Student's t test. Data are presented as mean ± SD. **p < 0.01; ***p < 0.001.B Correlation of serum SCT levels and serum triglycerides (TG), total cholesterol (TC) and nonesterified fatty acids (NEFA) levels in pregnant women in the middle trimester to the early third trimester (n = 15). Data information: Statistical significance was determined using Two-tailed Student's t test. Data are presented as mean ± SD. **p < 0.01; ***p < 0.001.C, D Plasma SCT levels of dams (C) at gestational day 18.5 (GD18.5) and embryo (D) at E18.5 (n = 8 per group). Data information: Statistical significance was determined using Two-tailed Student's t test. Data are presented as mean ± SD. **p < 0.01; ***p < 0.001.E Representative images of SCT immunohistochemical staining of duodenum from dams on standard (CON), whole grain (WG) and high fat (HFD) diet at GD18.5. SCT expression of WG is highest, but lower in HFD compared with CON. Scale bar, 50 μm (n = 10 per group). Right part shows quantification of SCT positive cells in duodenum from dams of three groups. Data information: Statistical significance was determined using Two-tailed Student's t test. Data are presented as mean ± SD. **p < 0.01; ***p < 0.001.


Figure 2B Change of body weight of M - SCT floxed and M - SCT KO mice upon HFD feeding ( n = 8 per group ) . Right part shows representative photograph of M - SCT floxed and M - SCT KO mice with HFD . Data information : Statistical significance was determined using Two - tailed Student ' s t test . C The weight of different tissues from M - SCT floxed and M - SCT KO mice with HFD ( n = 8 per group ) . Data information : Statistical significance was determined using Two - tailed Student ' s t test . Data are presented as mean ± SD . * * p < 0 . 01 ; * * * p < 0 . 001 ; ns , not statistically significant . D Random blood glucose levels in M - SCT floxed and M - SCT KO mice with HFD ( n = 8 per group ) . E Insulin levels in M - SCT floxed and M - SCT KO mice with HFD ( n = 8 per group ) . Data information : Statistical significance was determined using Two - tailed Student ' s t test . Data are presented as mean ± SD . * * p < 0 . 01 ; * * * p < 0 . 001 ; ns , not statistically significant . F Intraperitoneal glucose tolerance test ( GTT ) ( 1 . 0 g glucose / kg body weight ) in M - SCT floxed and M - SCT KO mice with HFD ( n = 5 per group ) and AUCs were calculated . Mice were fasted for 18h before assay . G Intraperitoneal insulin tolerance test ( ITT ) ( 0 . 75 U insulin / kg body weight ) in M - SCT floxed and M - SCT KO mice with HFD ( n = 5 per group ) and AUCs were calculated . mice were fasted for 6h before assays . Data information : Statistical significance was determined using Two - tailed Student ' s t test . Data are presented as mean ± SD . * * p < 0 . 01 ; * * * p < 0 . 001 ; ns , not statistically significant . H Serum TG , TC and NEFA levels in M - SCT floxed and M - SCT KO mice with HFD ( n = 8 per group ) . Data information : Statistical significance was determined using Two - tailed Student ' s t test . Data are presented as mean ± SD . * * p < 0 . 01 ; * * * p < 0 . 001 ; ns , not statistically significant .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2B Change of body weight of M-SCT floxed and M-SCT KO mice upon HFD feeding (n = 8 per group). Right part shows representative photograph of M-SCT floxed and M-SCT KO mice with HFD. Data information: Statistical significance was determined using Two-tailed Student's t test.C The weight of different tissues from M-SCT floxed and M-SCT KO mice with HFD (n = 8 per group). Data information: Statistical significance was determined using Two-tailed Student's t test. Data are presented as mean ± SD. **p < 0.01; ***p < 0.001; ns, not statistically significant.D Random blood glucose levels in M-SCT floxed and M-SCT KO mice with HFD (n = 8 per group). E Insulin levels in M-SCT floxed and M-SCT KO mice with HFD (n = 8 per group). Data information: Statistical significance was determined using Two-tailed Student's t test. Data are presented as mean ± SD. **p < 0.01; ***p < 0.001; ns, not statistically significant.F Intraperitoneal glucose tolerance test (GTT) (1.0 g glucose/ kg body weight) in M-SCT floxed and M-SCT KO mice with HFD (n = 5 per group) and AUCs were calculated. Mice were fasted for 18h before assay. G Intraperitoneal insulin tolerance test (ITT) (0.75 U insulin/kg body weight) in M-SCT floxed and M-SCT KO mice with HFD (n = 5 per group) and AUCs were calculated. mice were fasted for 6h before assays. Data information: Statistical significance was determined using Two-tailed Student's t test. Data are presented as mean ± SD. **p < 0.01; ***p < 0.001; ns, not statistically significant.H Serum TG, TC and NEFA levels in M-SCT floxed and M-SCT KO mice with HFD (n = 8 per group). Data information: Statistical significance was determined using Two-tailed Student's t test. Data are presented as mean ± SD. **p < 0.01; ***p < 0.001; ns, not statistically significant.


Figure 3A , B Time - resolved oxygen consumption ( A ) and heat production ( B ) in 48 - hour light / dark cycle measured by CLAMS in M - SCT floxed and M - SCT KO mice with HFD ( n = 6 per group ) , dark phase is marked as dark background . Regression - based absolute oxygen consumption ( A ) or heat production ( B ) analysis of body weight are shown in the right part . Data information : Statistical significance was determined using Two - tailed Student ' s t test . Data in panel A and B have been analyzed using ANCOVA with oxygen consumption or heat production as dependent variable , group as fixed variable and body weight as covariate . Data are presented as mean ± SD . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; ns , not statistically significant . C Body temperature of 8 - week - old M - SCT floxed and M - SCT KO male offspring with chow diet during cold exposure ( 4 ℃ ; n = 8 ) . Data information : Statistical significance was determined using Two - tailed Student ' s t test . Data are presented as mean ± SD . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; ns , not statistically significant . D Representative HE staining and UCP1 staining of Ing from M - SCT floxed and M - SCT KO mice with chow diet maintained at RT or 3 days of cold exposure ( 4 ℃ ) . Scale bar , 100 μm . E Relative mRNA levels of UCP1 , CIDEA , PRDM16 , PGC1α and DIO2 in iWAT from M - SCT floxed and M - SCT KO mice with chow diet ( n = 8 per group ) . Data information : Statistical significance was determined using Two - tailed Student ' s t test . Data are presented as mean ± SD . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; ns , not statistically significant . F Representative western blots of UCP1 , PRDM16 , PGC1α and HSP90 in iWAT from M - SCT floxed and M - SCT KO mice with chow diet after 3 days of cold exposure ( 4 ℃ ) . G Total UCP1 protein per mg iWAT of M - SCT floxed and M - SCT KO mice with chow diet maintained at RT or exposed to cold for 3 d ( 4 ℃ ) ( n = 3 ) . Data information : Statistical significance was determined using Two - tailed Student ' s t test . Data are presented as mean ± SD . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; ns , not statistically significant . H Relative mRNA levels of UCP1 , PGC1α , PRDM16 , CIDEA and DIO2 in SVF isolated from M - SCT KO and M - SCT floxed mice with HFD ( n = 8 per group ) . Data information : Statistical significance was determined using Two - tailed Student ' s t test . Data are presented as mean ± SD . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; ns , not statistically significant . I , J OCR of Oligomycin , FCCP and Antimycin A / Rotenone - treated matured adipocytes derived from iWAT - SVF of M - SCT KO and M - SCT floxed mice with HFD , and the AUC of OCR as indicated were calculated ( n = 5 per group ) . Data information : Statistical significance was determined using Two - tailed Student ' s t test . Data are presented as mean ± SD . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; ns , not statistically significant . K Glucose oxidation in adipocytes derived from iWAT - SVF of M - SCT KO and M - SCT floxed mice with HFD ( n = 8 per group ) . Data information : Statistical significance was determined using Two - tailed Student ' s t test . Data are presented as mean ± SD . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; ns , not statistically significant .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A, B Time-resolved oxygen consumption (A) and heat production (B) in 48-hour light/dark cycle measured by CLAMS in M-SCT floxed and M-SCT KO mice with HFD (n = 6 per group), dark phase is marked as dark background. Regression-based absolute oxygen consumption (A) or heat production (B) analysis of body weight are shown in the right part. Data information: Statistical significance was determined using Two-tailed Student's t test. Data in panel A and B have been analyzed using ANCOVA with oxygen consumption or heat production as dependent variable, group as fixed variable and body weight as covariate. Data are presented as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001; ns, not statistically significant.C Body temperature of 8-week-old M-SCT floxed and M-SCT KO male offspring with chow diet during cold exposure (4℃; n = 8). Data information: Statistical significance was determined using Two-tailed Student's t test. Data are presented as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001; ns, not statistically significant.D Representative HE staining and UCP1 staining of Ing from M-SCT floxed and M-SCT KO mice with chow diet maintained at RT or 3 days of cold exposure (4℃). Scale bar, 100 μm.E Relative mRNA levels of UCP1, CIDEA, PRDM16, PGC1α and DIO2 in iWAT from M-SCT floxed and M-SCT KO mice with chow diet (n = 8 per group). Data information: Statistical significance was determined using Two-tailed Student's t test. Data are presented as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001; ns, not statistically significant.F Representative western blots of UCP1, PRDM16, PGC1α and HSP90 in iWAT from M-SCT floxed and M-SCT KO mice with chow diet after 3 days of cold exposure (4℃). G Total UCP1 protein per mg iWAT of M-SCT floxed and M-SCT KO mice with chow diet maintained at RT or exposed to cold for 3 d (4℃) (n = 3). Data information: Statistical significance was determined using Two-tailed Student's t test. Data are presented as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001; ns, not statistically significant.H Relative mRNA levels of UCP1, PGC1α, PRDM16, CIDEA and DIO2 in SVF isolated from M-SCT KO and M-SCT floxed mice with HFD (n = 8 per group). Data information: Statistical significance was determined using Two-tailed Student's t test. Data are presented as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001; ns, not statistically significant.I, J OCR of Oligomycin, FCCP and Antimycin A/Rotenone-treated matured adipocytes derived from iWAT-SVF of M-SCT KO and M-SCT floxed mice with HFD, and the AUC of OCR as indicated were calculated (n = 5 per group). Data information: Statistical significance was determined using Two-tailed Student's t test. Data are presented as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001; ns, not statistically significant.K Glucose oxidation in adipocytes derived from iWAT-SVF of M-SCT KO and M-SCT floxed mice with HFD (n = 8 per group). Data information: Statistical significance was determined using Two-tailed Student's t test. Data are presented as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001; ns, not statistically significant.


Figure 4B Stacked bars showing the Proportional distribution of hypo - and hypermethylated DMRs ( M - SCT KO vs M - SCT floxed ) in different regions . Data information : Statistical significance was determined using Two - tailed Student ' s t test . Data are presented as mean ± SD . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; ns , not statistically significant . C Heat map of DMR methylation level in mRNA between M - SCT floxed and M - SCT KO . The color bar indicates the normalized tag counts in M - SCT floxed or M - SCT KO . Positive z scores , shown in the table , indicate hypermethylated regions , while negative z scores imply hypomethylated regions . D As in ( C ) , for enhancer region . E Scatter plot representation of the most significant difference of methylation in mRNA ( left ) and enhancer ( right ) regions between M - SCT floxed and M - SCT KO ( p < 0 . 005 ) . Data information : Statistical significance was determined using Two - tailed Student ' s t test . F Volcano plot DNA methylation represents all DMRs in mRNA ( left ) and enhancer ( right ) regions . Cutoffs ( vertical and horizontal dashed lines ) used to identify DMRs between M - SCT floxed and M - SCT KO mice . The vertical lines correspond to twofold up and down , respectively , and the horizontal line represents a P value of 0 . 01 . Data information : Statistical significance was determined using Two - tailed Student ' s t test . I , J Schematic diagram showing the CpG islands of Mus musculus PRDM16 ( I ) and CIDEA ( J ) genes . The conditions to be met by CpG islands identified were : 200 - 300 bp length , CG content of > 50 % and observed CpG content vs expected CpG content > 0 . 6 . Methylation levels of each CpG islands in PRDM16 ( I ) and CIDEA ( J ) are presented as the fold change relative to the average value of the M - SCT floxed group and β - actin promoter was used as a negative control ( n = 3 per group ) . Data information : Statistical significance was determined using Two - tailed Student ' s t test . Data are presented as mean ± SD . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; ns , not statistically significant .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4B Stacked bars showing the Proportional distribution of hypo- and hypermethylated DMRs (M-SCT KO vs M-SCT floxed) in different regions. Data information: Statistical significance was determined using Two-tailed Student's t test. Data are presented as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001; ns, not statistically significant.C Heat map of DMR methylation level in mRNA between M-SCT floxed and M-SCT KO. The color bar indicates the normalized tag counts in M-SCT floxed or M-SCT KO. Positive z scores, shown in the table, indicate hypermethylated regions, while negative z scores imply hypomethylated regions. D As in (C), for enhancer region.E Scatter plot representation of the most significant difference of methylation in mRNA (left) and enhancer (right) regions between M-SCT floxed and M-SCT KO (p < 0.005). Data information: Statistical significance was determined using Two-tailed Student's t test.F Volcano plot DNA methylation represents all DMRs in mRNA (left) and enhancer (right) regions. Cutoffs (vertical and horizontal dashed lines) used to identify DMRs between M-SCT floxed and M-SCT KO mice. The vertical lines correspond to twofold up and down, respectively, and the horizontal line represents a P value of 0.01. Data information: Statistical significance was determined using Two-tailed Student's t test.I, J Schematic diagram showing the CpG islands of Mus musculus PRDM16 (I) and CIDEA (J) genes. The conditions to be met by CpG islands identified were: 200-300 bp length, CG content of > 50% and observed CpG content vs expected CpG content > 0.6. Methylation levels of each CpG islands in PRDM16 (I) and CIDEA (J) are presented as the fold change relative to the average value of the M-SCT floxed group and β-actin promoter was used as a negative control (n = 3 per group). Data information: Statistical significance was determined using Two-tailed Student's t test. Data are presented as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001; ns, not statistically significant.


Figure 5A Western blots analysis of DNMT1 , DNMT3a , DNMT3b and HSP90 in Ing from M - SCT KO and M - SCT floxed mice with HFD . B Relative mRNA levels of DNMT1 , DNMT3a and DNMT3b in Ing from M - SCT KO and M - SCT floxed mice with HFD ( n = 8 ) . Statistical significance was determined using Two - tailed Student ' s t test . Data are presented as mean ± SD . ns , not statistically significant . C Western blots analysis of p - AMPKα , AMPKα , p - ACC , ACC , DNMT1 and HSP90 in embryo of M - SCT KO and M - SCT floxed mice at E18 . 5 . D Western blot analysis of p - AMPKα , AMPKα , p - ACC , ACC , DNMT1 and HSP90 in mouse embryo fibroblast ( MEF ) cells treated with 10 nM or 100 nM SCT . E Western blot analysis of p - AMPKα , AMPKα , p - ACC , ACC , DNMT1 and HSP90 in MEF cells treated with 10 nM SCT or 20 μM compound C ( C . C . ) for 24 h . F Western blot analysis of p - AMPKα , AMPKα , DNMT1 , SCTR and HSP90 . MEF cells were transfected with indicated siRNA and then treated with 1 mM AMPK agonist ( AICAR ) for 24 h . G Western blot analysis of p - AMPKα , AMPKα , p - ACC , ACC , DNMT1 and HSP90 in AMPK + / + or AMPKα1 / α2 double knockout ( AMPK - / - ) MEF cells treated with 10 nM SCT for 24 h . H , I Western blot analysis of Flag - DNMT1 in Flag - DNMT1 - transfected HEK293T cells treated with AICAR with indicated dose or time periods . J HEK293T cells were transfected with indicated plasmids and then treated with 10 nM SCT for 24 h , followed by IP and IB analysis . K HEK293T cells were transfected with indicated plasmids or siRNA and then lysed , followed by IP and IB analysis . L AMPK + / + or AMPK - / - MEF cells were transfected with indicated plasmids and then lysed , followed by IP and IB analysis .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A Western blots analysis of DNMT1, DNMT3a, DNMT3b and HSP90 in Ing from M-SCT KO and M-SCT floxed mice with HFD.B Relative mRNA levels of DNMT1, DNMT3a and DNMT3b in Ing from M-SCT KO and M-SCT floxed mice with HFD (n = 8). Statistical significance was determined using Two-tailed Student's t test. Data are presented as mean ± SD. ns, not statistically significant.C Western blots analysis of p-AMPKα, AMPKα, p-ACC, ACC, DNMT1 and HSP90 in embryo of M-SCT KO and M-SCT floxed mice at E18.5.D Western blot analysis of p-AMPKα, AMPKα, p-ACC, ACC, DNMT1 and HSP90 in mouse embryo fibroblast (MEF) cells treated with 10 nM or 100 nM SCT.E Western blot analysis of p-AMPKα, AMPKα, p-ACC, ACC, DNMT1 and HSP90 in MEF cells treated with 10 nM SCT or 20 μM compound C (C.C.) for 24 h.F Western blot analysis of p-AMPKα, AMPKα, DNMT1, SCTR and HSP90. MEF cells were transfected with indicated siRNA and then treated with 1 mM AMPK agonist (AICAR) for 24 h.G Western blot analysis of p-AMPKα, AMPKα, p-ACC, ACC, DNMT1 and HSP90 in AMPK+/+ or AMPKα1/α2 double knockout (AMPK-/-) MEF cells treated with 10 nM SCT for 24 h.H, I Western blot analysis of Flag-DNMT1 in Flag-DNMT1-transfected HEK293T cells treated with AICAR with indicated dose or time periods.J HEK293T cells were transfected with indicated plasmids and then treated with 10 nM SCT for 24 h, followed by IP and IB analysis.K HEK293T cells were transfected with indicated plasmids or siRNA and then lysed, followed by IP and IB analysis.L AMPK+/+ or AMPK-/- MEF cells were transfected with indicated plasmids and then lysed, followed by IP and IB analysis.


Figure 6A Serum SCT levels in M - HFD and M - HFD + SCT mice with HFD ( n = 8 per group ) . Data information : Statistical significance was determined using Two - tailed Student ' s t test . Data are presented as mean ± SD . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 . B Change of body weight of M - HFD ( offspring of dams with high - fat pregnant diets ) and M - HFD + SCT ( offspring of dams with high - fat pregnant diets supplied with SCT ) mice upon HFD feeding ( n = 8 per group ) . Representative photograph of M - HFD and M - HFD + SCT mice after HFD feeding . Data information : Statistical significance was determined using Two - tailed Student ' s t test . Data are presented as mean ± SD . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 . C , Time - resolved oxygen consumption ( C ) in 48 - hour light / dark cycle measured by CLAMS in M - HFD and M - HFD + SCT mice with HFD ( n = 5 per group ) , dark phase is marked as dark background . Regression - based absolute oxygen consumption ( C ) analysis of body weight are shown in the right part . Data information : Statistical significance was determined using Two - tailed Student ' s t test . Data in panel C have been analyzed using ANCOVA with oxygen consumption or heat production as dependent variable , group as fixed variable and body weight as covariate . Data are presented as mean ± SD . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 . D Time - resolved heat production ( D ) in 48 - hour light / dark cycle measured by CLAMS in M - HFD and M - HFD + SCT mice with HFD ( n = 5 per group ) , dark phase is marked as dark background . Regression - based absolute heat production ( D ) analysis of body weight are shown in the right part . Data information : Statistical significance was determined using Two - tailed Student ' s t test . Data in panel D have been analyzed using ANCOVA with oxygen consumption or heat production as dependent variable , group as fixed variable and body weight as covariate . Data are presented as mean ± SD . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 . E GTT ( 1 g glucose / kg body weight ) in M - HFD and M - HFD + SCT mice with HFD ( n = 5 per group ) and AUCs were calculated in right part . Mice were fasted for 18h before assays . Data information : Statistical significance was determined using Two - tailed Student ' s t test . Data are presented as mean ± SD . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 . F ITT ( 0 . 75 U insulin / kg body weight ) in M - HFD and M - HFD + SCT mice with HFD , and AUCs were calculated in bottom ( n = 5 per group ) . Mice were fasted for 6h before assays . Data information : Statistical significance was determined using Two - tailed Student ' s t test . Data are presented as mean ± SD . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 . G Serum TG , TC and NEFA levels in M - HFD and M - HFD + SCT mice with HFD . ( n = 8 per group ) . Data information : Statistical significance was determined using Two - tailed Student ' s t test . Data are presented as mean ± SD . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 . H Representative HE staining and UCP1 staining of iWAT from M - HFD and M - HFD + SCT mice with HFD . Scale bar , 100 μm ( n = 5 per group ) . I Relative mRNA levels of UCP1 , PGC1α , PRDM16 , CIDEA and DIO2 in iWAT from M - HFD and M - HFD + SCT mice with HFD ( n = 5 per group ) . Data information : Statistical significance was determined using Two - tailed Student ' s t test . Data are presented as mean ± SD . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6A Serum SCT levels in M-HFD and M-HFD+SCT mice with HFD (n = 8 per group). Data information: Statistical significance was determined using Two-tailed Student's t test. Data are presented as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001.B Change of body weight of M-HFD (offspring of dams with high-fat pregnant diets) and M-HFD+SCT (offspring of dams with high-fat pregnant diets supplied with SCT) mice upon HFD feeding (n = 8 per group). Representative photograph of M-HFD and M-HFD+SCT mice after HFD feeding. Data information: Statistical significance was determined using Two-tailed Student's t test. Data are presented as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001.C, Time-resolved oxygen consumption (C) in 48-hour light/dark cycle measured by CLAMS in M-HFD and M-HFD+SCT mice with HFD (n = 5 per group), dark phase is marked as dark background. Regression-based absolute oxygen consumption (C) analysis of body weight are shown in the right part. Data information: Statistical significance was determined using Two-tailed Student's t test. Data in panel C have been analyzed using ANCOVA with oxygen consumption or heat production as dependent variable, group as fixed variable and body weight as covariate. Data are presented as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001.D Time-resolved heat production (D) in 48-hour light/dark cycle measured by CLAMS in M-HFD and M-HFD+SCT mice with HFD (n = 5 per group), dark phase is marked as dark background. Regression-based absolute heat production (D) analysis of body weight are shown in the right part. Data information: Statistical significance was determined using Two-tailed Student's t test. Data in panel D have been analyzed using ANCOVA with oxygen consumption or heat production as dependent variable, group as fixed variable and body weight as covariate. Data are presented as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001.E GTT (1 g glucose/kg body weight) in M-HFD and M-HFD+SCT mice with HFD (n = 5 per group) and AUCs were calculated in right part. Mice were fasted for 18h before assays. Data information: Statistical significance was determined using Two-tailed Student's t test. Data are presented as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001.F ITT (0.75 U insulin/kg body weight) in M-HFD and M-HFD+SCT mice with HFD, and AUCs were calculated in bottom (n = 5 per group). Mice were fasted for 6h before assays. Data information: Statistical significance was determined using Two-tailed Student's t test. Data are presented as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001.G Serum TG, TC and NEFA levels in M-HFD and M-HFD+SCT mice with HFD. (n = 8 per group). Data information: Statistical significance was determined using Two-tailed Student's t test. Data are presented as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001.H Representative HE staining and UCP1 staining of iWAT from M-HFD and M-HFD+SCT mice with HFD. Scale bar, 100 μm (n = 5 per group).I Relative mRNA levels of UCP1, PGC1α, PRDM16, CIDEA and DIO2 in iWAT from M-HFD and M-HFD+SCT mice with HFD (n = 5 per group). Data information: Statistical significance was determined using Two-tailed Student's t test. Data are presented as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001.


Figure 1HeLa cells co - transfected with EMTB - CFP - FRB and YFP - FKBP were treated with 100 nM rapamycin ( Rapa ) . Addition of rapamycin rapidly translocated YFP - FKBP onto EMTB - CFP - FRB - labeled microtubules and increased the FRET signal . The scale for the FRET / CFP intensity ratio is shown . Scale bar , 10 μm . The normalized intensity of FRET / CFP in cells before and after rapamycin ( blue ) and 0 . 1 % DMSO ( control ; red ) treatment . n = 6 and 10 cells in the rapamycin and DMSO groups , respectively , from three independent experiments . Data are shown as the mean ± S . E . M . HeLa cells co - transfected with the indicated constructs were treated with nocodazole ( Noc , 3 . 3 µM ; left ) or rapamycin ( Rapa , 100 nM ; right ) for 0 , 30 , and 60 min . Transfected cells were then fixed and labeled with anti - acetylated tubulin ( red ) . Dotted lines indicate the boundary of transfected cells . Scale bars , 10 μm . The intensity of acetylated tubulin in cells transfected with EMTB - CFP - FRB ( orange ) or cotransfected with EMTB - CFP - FRB and dNSpastin3Q - YFP - FKBP ( blue ) after addition of nocodazole ( Noc , 3 . 3 µM ) or rapamycin ( Rapa , 100 nM ) for the indicated times . n = 123 and 81 cells in the nocodazole and dNSpastin3Q groups , respectively , from three independent experiments . Data are shown as the mean ± S . E . M . Student ' s t - tests were performed , with p - values indicated .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1HeLa cells co-transfected with EMTB-CFP-FRB and YFP-FKBP were treated with 100 nM rapamycin (Rapa). Addition of rapamycin rapidly translocated YFP-FKBP onto EMTB-CFP-FRB-labeled microtubules and increased the FRET signal. The scale for the FRET/CFP intensity ratio is shown. Scale bar, 10 μm. The normalized intensity of FRET/CFP in cells before and after rapamycin (blue) and 0.1% DMSO (control; red) treatment. n = 6 and 10 cells in the rapamycin and DMSO groups, respectively, from three independent experiments. Data are shown as the mean ± S.E.M.HeLa cells co-transfected with the indicated constructs were treated with nocodazole (Noc, 3.3 µM; left) or rapamycin (Rapa, 100 nM; right) for 0, 30, and 60 min. Transfected cells were then fixed and labeled with anti-acetylated tubulin (red). Dotted lines indicate the boundary of transfected cells. Scale bars, 10 μm. The intensity of acetylated tubulin in cells transfected with EMTB-CFP-FRB (orange) or cotransfected with EMTB-CFP-FRB and dNSpastin3Q-YFP-FKBP (blue) after addition of nocodazole (Noc, 3.3 µM) or rapamycin (Rapa, 100 nM) for the indicated times. n = 123 and 81 cells in the nocodazole and dNSpastin3Q groups, respectively, from three independent experiments. Data are shown as the mean ± S.E.M. Student's t-tests were performed, with p-values indicated.


Figure 2NIH3T3 fibroblasts co - transfected with 5HT6 - mCherry ( 5HT6 - mCh ; a ciliary membrane marker ; red ) , CFP - FRB - MAP4m ( blue ) , and dNSpastin3Q - YFP - FKBP ( green ) were serum - starved for 24 hr to induce ciliogenesis . Ciliated cells were then treated with rapamycin ( Rapa , 100 nM ) to induce dNSpastin3Q - YFP - FKBP recruitment to axonemal microtubules . Dotted lines indicate the cell boundaries . Scale bar , 5 μm . The normalized length of axonemes ( blue ) , primary cilia ( red ) , and dNSpastin3Q - YFP - FKBP in cilia ( green ) upon rapamycin treatment . n = 6 cells from three independent experiments . Data are shown as the mean ± S . E . M . HeLa cells co - transfected with H2B - mCherry ( H2B - mCh ; a chromosome marker ; red ) , CFP - FRB - MAP4m ( a marker of mitotic spindles ; blue ) , and dNSpastin3Q - YFP - FKBP ( green ) were synchronized in metaphase and treated with rapamycin ( 100 nM ) to induce dNSpastin3Q - YFP - FKBP recruitment to mitotic spindles . Dotted lines indicate the cell boundaries . Scale bar , 10 μm . The normalized area of the mitotic spindle ( blue ) and intensity of dNSpastin3Q - YFP - FKBP in the mitotic spindle ( green ) upon rapamycin treatment . n = 6 cells from three independent experiments . Data are shown as the mean ± S . E . M . HeLa cells co - transfected with H2B - mCherry ( red ) , CFP - FRB - MAP4m ( intercellular bridges ; blue ) , and dNSpastin3Q - YFP - FKBP ( green ) were treated with rapamycin ( 100 nM ) to induce dNSpastin3Q - YFP - FKBP recruitment to intercellular bridges . Arrows indicate the intercellular bridges . Dotted lines indicate the cell boundaries . Scale bar , 10 μm . The normalized area of intercellular bridges ( blue ) and intensity of dNSpastin3Q - YFP - FKBP at intercellular bridges ( green ) upon rapamycin treatment . n = 5 cells from three independent experiments . Data are shown as the mean ± S . E . M .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2NIH3T3 fibroblasts co-transfected with 5HT6-mCherry (5HT6-mCh; a ciliary membrane marker; red), CFP-FRB-MAP4m (blue), and dNSpastin3Q-YFP-FKBP (green) were serum-starved for 24 hr to induce ciliogenesis. Ciliated cells were then treated with rapamycin (Rapa, 100 nM) to induce dNSpastin3Q-YFP-FKBP recruitment to axonemal microtubules. Dotted lines indicate the cell boundaries. Scale bar, 5 μm. The normalized length of axonemes (blue), primary cilia (red), and dNSpastin3Q-YFP-FKBP in cilia (green) upon rapamycin treatment. n = 6 cells from three independent experiments. Data are shown as the mean ± S.E.M.HeLa cells co-transfected with H2B-mCherry (H2B-mCh; a chromosome marker; red), CFP-FRB-MAP4m (a marker of mitotic spindles; blue), and dNSpastin3Q-YFP-FKBP (green) were synchronized in metaphase and treated with rapamycin (100 nM) to induce dNSpastin3Q-YFP-FKBP recruitment to mitotic spindles. Dotted lines indicate the cell boundaries. Scale bar, 10 μm. The normalized area of the mitotic spindle (blue) and intensity of dNSpastin3Q-YFP-FKBP in the mitotic spindle (green) upon rapamycin treatment. n = 6 cells from three independent experiments. Data are shown as the mean ± S.E.M.HeLa cells co-transfected with H2B-mCherry (red), CFP-FRB-MAP4m (intercellular bridges; blue), and dNSpastin3Q-YFP-FKBP (green) were treated with rapamycin (100 nM) to induce dNSpastin3Q-YFP-FKBP recruitment to intercellular bridges. Arrows indicate the intercellular bridges. Dotted lines indicate the cell boundaries. Scale bar, 10 μm.   The normalized area of intercellular bridges (blue) and intensity of dNSpastin3Q-YFP-FKBP at intercellular bridges (green) upon rapamycin treatment. n = 5 cells from three independent experiments. Data are shown as the mean ± S.E.M.


Figure 3COS7 cells transfected with TagRFP - FRB - A1AY1 ( red ) were labeled by anti - α - tubulin antibody ( green ; upper panel ) , anti - tyrosinated tubulin antibody ( green ; middle panel ) , or anti - detyrosinated tubulin antibody ( green ; lower panel ) , respectively . Scale bar , 10 µm . The intensity profiles of TagRFP - FRB - A1AY1 ( red ) and tubulin with the indicated PTMs ( green ) along dotted lines drawn in ( A ) . The Pearson ' s correlation coefficients for TagRFP - FRB - A1AY1 and indicated tubulin ( tub ) subtypes were calculated , and data are shown as the mean ± S . E . M . n = 16 , 13 , and 15 cells from left to right , respectively ; three independent experiments . COS7 cells co - transfected with TagRFP - FRB - A1AY1 ( red ) and TagCFP - FKBP ( green ) were treated with rapamycin ( 100 nM ) . The addition of rapamycin rapidly recruits TagCFP - FKBP from cytosol onto A1AY1 - labeled microtubules ( arrows ) . Scale bar , 10 µm . The normalized intensity of TagCFP - FKBP at A1AY1 - labeled microtubules upon rapamycin treatments . n = 7 cells from three independent experiments . Data are shown as the mean ± S . E . M . COS7 cells co - transfected with TagRFP - FRB - A1AY1 ( red ) and dNSpastin3Q - TagCFP - FKBP ( cyan ) were treated with 0 . 1 % DMSO or rapamycin ( 100 nM ) for 1 hr and followed by immunostaining with anti - tyrosinated tubulin antibody ( green ) . Dotted lines highlight the transfected cells . Scale bar , 20 μm . The normalized intensity of tyrosinated microtubules in TagRFP - FRB - A1AY1 and dNSpastin3Q - TagCFP - FKBP co - transfected cells upon 0 . 1 % DMSO or rapamycin treatment for 1 hr . n = 31 and 26 cells in DMSO and rapamycin treated groups , respectively , from three independent experiments . Data ( blue ) represent as mean ± S . E . M . Student ' s t - tests were performed with p values indicated .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3COS7 cells transfected with TagRFP-FRB-A1AY1 (red) were labeled by anti-α-tubulin antibody (green; upper panel), anti-tyrosinated tubulin antibody (green; middle panel), or anti-detyrosinated tubulin antibody (green; lower panel), respectively. Scale bar, 10 µm. The intensity profiles of TagRFP-FRB-A1AY1 (red) and tubulin with the indicated PTMs (green) along dotted lines drawn in (A). The Pearson's correlation coefficients for TagRFP-FRB-A1AY1 and indicated tubulin (tub) subtypes were calculated, and data are shown as the mean ± S.E.M. n = 16, 13, and 15 cells from left to right, respectively; three independent experiments.COS7 cells co-transfected with TagRFP-FRB-A1AY1 (red) and TagCFP-FKBP (green) were treated with rapamycin (100 nM). The addition of rapamycin rapidly recruits TagCFP-FKBP from cytosol onto A1AY1-labeled microtubules (arrows). Scale bar, 10 µm. The normalized intensity of TagCFP-FKBP at A1AY1-labeled microtubules upon rapamycin treatments. n=7 cells from three independent experiments. Data are shown as the mean ± S.E.M.COS7 cells co-transfected with TagRFP-FRB-A1AY1 (red) and dNSpastin3Q-TagCFP-FKBP (cyan) were treated with 0.1% DMSO or rapamycin (100 nM) for 1 hr and followed by immunostaining with anti-tyrosinated tubulin antibody (green). Dotted lines highlight the transfected cells. Scale bar, 20 μm. The normalized intensity of tyrosinated microtubules in TagRFP-FRB-A1AY1 and dNSpastin3Q-TagCFP-FKBP co-transfected cells upon 0.1% DMSO or rapamycin treatment for 1 hr. n=31 and 26 cells in DMSO and rapamycin treated groups, respectively, from three independent experiments. Data (blue) represent as mean ± S.E.M. Student's t-tests were performed with p values indicated.


Figure 4COS7 cells co - transfected with EMTB - YFP - CIBN and mCh - Cry2 ( red ) were incubated with SPY650 - tubulin ( SPY650 - tub ) to visualize microtubules . The cells were illuminated by blue light within a specified region ( indicated by the dotted circle ) for the indicated time period . Scale bar , 10 μm . The normalized intensity of mCh - Cry2 at microtubules in illuminated regions ( red ) and non - illuminated regions ( green ) . n = 6 cells from three independent experiments . Data are shown as the mean ± S . E . M . COS7 cells co - transfected with EMTB - YFP - CIBN and dNSpastin3Q - mCh - Cry2 were incubated with SPY650 - tubulin to visualize microtubules . The cells were illuminated by blue light as in ( A ) . Scale bar , 10 μm . The normalized intensity of dNSpastin3Q - mCh - Cry2 and mCh - Cry2 at microtubules and the normalized area of SPY650 - tubulin in illuminated regions and non - illuminated regions . n = 6 and 6 cells in dNSpastin3Q - mCh - Cry2 and mCh - Cry2 groups , respectively , from three independent experiments . Data are shown as the mean ± S . E . M .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4COS7 cells co-transfected with EMTB-YFP-CIBN and mCh-Cry2 (red) were incubated with SPY650-tubulin (SPY650-tub) to visualize microtubules. The cells were illuminated by blue light within a specified region (indicated by the dotted circle) for the indicated time period. Scale bar, 10 μm. The normalized intensity of mCh-Cry2 at microtubules in illuminated regions (red) and non-illuminated regions (green). n = 6 cells from three independent experiments. Data are shown as the mean ± S.E.M.COS7 cells co-transfected with EMTB-YFP-CIBN and dNSpastin3Q-mCh-Cry2 were incubated with SPY650-tubulin to visualize microtubules. The cells were illuminated by blue light as in (A). Scale bar, 10 μm. The normalized intensity of dNSpastin3Q-mCh-Cry2 and mCh-Cry2 at microtubules and the normalized area of SPY650-tubulin in illuminated regions and non-illuminated regions. n = 6 and 6 cells in dNSpastin3Q-mCh-Cry2 and mCh-Cry2 groups, respectively, from three independent experiments. Data are shown as the mean ± S.E.M.


Figure 5E - F . COS7 cells co - transfected with EMTB - CFP - FRB , dNSpastin3Q - mCh - FKBP , and Tom20 - Neon were treated with rapamycin ( 100 nM ) for acute microtubule disruption . The morphology of mitochondria at different levels of microtubule disruption is shown . Scale bar , 10 µm . The ratio of the long axis to the short axis of each mitochondrion and microtubule filament area was determined . n = 4 different cells , three independent experiments . Data are shown as the mean ± S . E . M . Data information : Student ' s t - tests were performed with p values indicated . G - H . The normalized intensity of MitoTracker Red in non - transfected cells ( Mock ) or cells co - transfected with EMTB - CFRP - FRB and dNSpastin3Q - YFP - FKBP or dNSpastin3QED - YFP - FKBP after rapamycin ( Rapa , 100 nM ) treatment for the indicated time . Scale bar , 10 µm . n = 41 , 6 , and 18 cells from three independent experiments . Data are shown as the mean ± S . E . M . Data information : Student ' s t - tests were performed with p values indicated .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5E-F. COS7 cells co-transfected with EMTB-CFP-FRB, dNSpastin3Q-mCh-FKBP, and Tom20-Neon were treated with rapamycin (100 nM) for acute microtubule disruption. The morphology of mitochondria at different levels of microtubule disruption is shown. Scale bar, 10 µm. The ratio of the long axis to the short axis of each mitochondrion and microtubule filament area was determined. n= 4 different cells, three independent experiments. Data are shown as the mean ± S.E.M. Data information: Student's t-tests were performed with p values indicated.G-H. The normalized intensity of MitoTracker Red in non-transfected cells (Mock) or cells co-transfected with EMTB-CFRP-FRB and dNSpastin3Q-YFP-FKBP or dNSpastin3QED-YFP-FKBP after rapamycin (Rapa, 100 nM) treatment for the indicated time. Scale bar, 10 µm. n = 41, 6, and 18 cells from three independent experiments. Data are shown as the mean ± S.E.M. Data information: Student's t-tests were performed with p values indicated.


Figure 6COS7 cells co - transfected with EMTB - CFP - FRB ( blue ) , dNSpastin3Q - mCh - FKBP ( red ) , and Lifeact - Neon ( heatmap and green ) were treated with rapamycin ( Rapa , 100 nM ) to induce microtubule disassembly . Arrows and arrowheads indicate lamellipodia and stress fibers , respectively . Insets show images of lamellipodia morphology from regions corresponding to the dotted squares with overexposed CFP and mCherry signals . Scale bar , 10 µm . The normalized level of microtubule area , lamellipodia length , and filopodia density of COS - 7 cells treated as in ( A ) . n = 4 cells from three independent experiments . Data are shown as the mean ± S . E . M .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6COS7 cells co-transfected with EMTB-CFP-FRB (blue), dNSpastin3Q-mCh-FKBP (red), and Lifeact-Neon (heatmap and green) were treated with rapamycin (Rapa, 100 nM) to induce microtubule disassembly. Arrows and arrowheads indicate lamellipodia and stress fibers, respectively. Insets show images of lamellipodia morphology from regions corresponding to the dotted squares with overexposed CFP and mCherry signals. Scale bar, 10 µm. The normalized level of microtubule area, lamellipodia length, and filopodia density of COS-7 cells treated as in (A). n = 4 cells from three independent experiments. Data are shown as the mean ± S.E.M.


Figure 1 ( A ) Measurement of maximum glycolytic capacity of MOTS - c in dystrophic muscle cells after oligomycin and 2 - DG treatments ( n = 6 ; * p < 0 . 05 ) . ( B ) Quantification of ECAR changes in relative to basal ECAR in dystrophic muscle cells treated with MOTS - c ( n = 6 ; * p < 0 . 05 ) . ( C ) ATP assay for dystrophic muscle cells in the presence of MOTS - c ( n = 6 ; * p < 0 . 05 ) . NC refers to untreated H2K or H2K mdx cells . ( D ) Tissue distribution and quantitative analysis of Rhodamine B - labeled MOTS - c in mdx mice after single intravenous injection ( 500 μg ) ( n = 6 ; * p < 0 . 05 , * p < 0 . 001 ) . Tissues were harvested and examined 2 h after injection . This experiment was repeated twice . NC refers to untreated mdx mice . TA - tibialis anterior , Q - quadriceps , G - gastrocnemius , T - triceps , A - abdominal muscle , D - diaphragm , H - heart , Li - liver , S - spleen , Lu - lung and K - kidney . ( E ) Measurement of ATP levels in peripheral muscles of mdx mice after single intravenous injection of MOTS - c ( 500 μg ) ( n = 6 ; * p < 0 . 05 ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(A) Measurement of maximum glycolytic capacity of MOTS-c in dystrophic muscle cells after oligomycin and 2-DG treatments (n=6; *p<0.05). (B) Quantification of ECAR changes in relative to basal ECAR in dystrophic muscle cells treated with MOTS-c (n=6; *p<0.05).  (C) ATP assay for dystrophic muscle cells in the presence of MOTS-c (n=6; *p<0.05). NC refers to untreated H2K or H2K mdx cells.  (D) Tissue distribution and quantitative analysis of Rhodamine B-labeled MOTS-c in mdx mice after single intravenous injection (500 μg) (n=6; *p<0.05, *p<0.001). Tissues were harvested and examined 2 h after injection. This experiment was repeated twice. NC refers to untreated mdx mice. TA-tibialis anterior, Q-quadriceps, G-gastrocnemius, T-triceps, A-abdominal muscle, D-diaphragm, H-heart, Li-liver, S-spleen, Lu-lung and K-kidney.  (E) Measurement of ATP levels in peripheral muscles of mdx mice after single intravenous injection of MOTS-c (500 μg) (n=6; *p<0.05).


Figure 2 ( A ) Measurement of PMO uptake in dystrophic muscle cells ( H2K mdx cells ) in the presence of MOTS - c ( scale bar : 10 μm ) ( n = 6 ; * * p < 0 . 001 ) . PMO - M refers to PMO and MOTS - c ( the same is for the rest unless otherwise specified ) . ( B ) Tissue distribution of FITC - labeled PMO ( 50 mg / kg ) mixed with MOTS - c ( 500 μg ) in mdx mice 48 h after single intravenous injection . NC represents untreated mdx control . The abbreviation is the same as Figure 1D . Color scale represents the fluorescence intensity . ( C ) Quantification of fluorescence intensity in body - wide tissues of mdx mice treated with PMO - M or PMO alone ( n = 3 ; * p < 0 . 05 ) . The comparison was conducted between PMO - M and PMO . ( D , E ) Immunohistochemistry ( D ) and quantitative analysis ( E ) of dystrophin - positive fibers in mdx TA muscles treated with single intramuscular injection of 0 . 5 μg PMO mixed with MOTS - c ( 20 μg ) ( PMO - M ) or PMO alone ( scale bar : 100 μm ) ( n = 3 ; * * p < 0 . 001 ) . C57 means wild - type control C57BL / 6 . ( F ) Representative Western blot and quantitative analysis for dystrophin expression in TA muscles from mdx mice treated with single intramuscular injection of PMO - M or PMO alone ( n = 3 ; * p < 0 . 05 ) . 0 . 5 µg and 2 . 5 µg total protein from C57BL / 6 and 50 µg from muscle samples of untreated and treated mdx mice were loaded . α - actinin was used as the loading control . TA muscles from C57BL / 6 were used as normal controls ( the same is for all Western blots unless otherwise specified ) . ( G ) Systemic evaluation of MOTS - c ( 500 μg ) and PMO at the dose of 50 mg / kg / week for 3 weeks in adult mdx mice intravenously . Tissues were examined two weeks after last injection . Immunohistochemistry for dystrophin - positive fibers in body - wide muscles of treated mdx mice ( scale bar : 100 μm ) . C57 means wild - type control C57BL / 6 . ( H , I ) Western blot ( H ) and quantitative analysis ( I ) for dystrophin expression in body - wide muscles from treated mdx mice ( n = 3 ; * p < 0 . 05 ) . 0 . 5 μg , 2 . 5 μg , 5 μg and 10 μg total protein from C57BL / 6 and 20 μg of muscle samples from untreated and treated mdx mice were loaded . α - actinin was used as the loading control .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2 (A) Measurement of PMO uptake in dystrophic muscle cells (H2K mdx cells) in the presence of MOTS-c (scale bar: 10 μm) (n=6; **p<0.001). PMO-M refers to PMO and MOTS-c (the same is for the rest unless otherwise specified).  (B) Tissue distribution of FITC-labeled PMO (50 mg/kg) mixed with MOTS-c (500 μg) in mdx mice 48 h after single intravenous injection. NC represents untreated mdx control. The abbreviation is the same as Figure 1D. Color scale represents the fluorescence intensity. (C) Quantification of fluorescence intensity in body-wide tissues of mdx mice treated with PMO-M or PMO alone (n=3; *p<0.05). The comparison was conducted between PMO-M and PMO.  (D, E) Immunohistochemistry (D) and quantitative analysis (E) of dystrophin-positive fibers in mdx TA muscles treated with single intramuscular injection of 0.5 μg PMO mixed with MOTS-c (20 μg) (PMO-M) or PMO alone (scale bar: 100 μm) (n=3; **p<0.001). C57 means wild-type control C57BL/6.  (F) Representative Western blot and quantitative analysis for dystrophin expression in TA muscles from mdx mice treated with single intramuscular injection of PMO-M or PMO alone (n=3; *p<0.05). 0.5 µg and 2.5 µg total protein from C57BL/6 and 50 µg from muscle samples of untreated and treated mdx mice were loaded. α-actinin was used as the loading control. TA muscles from C57BL/6 were used as normal controls (the same is for all Western blots unless otherwise specified).  (G) Systemic evaluation of MOTS-c (500 μg) and PMO at the dose of 50 mg/kg/week for 3 weeks in adult mdx mice intravenously. Tissues were examined two weeks after last injection. Immunohistochemistry for dystrophin- positive fibers in body-wide muscles of treated mdx mice (scale bar: 100 μm). C57 means wild-type control C57BL/6.  (H, I) Western blot (H) and quantitative analysis (I) for dystrophin expression in body-wide muscles from treated mdx mice (n=3; *p<0.05). 0.5 μg, 2.5 μg, 5 μg and 10 μg total protein from C57BL/6 and 20 μg of muscle samples from untreated and treated mdx mice were loaded. α-actinin was used as the loading control.


Figure 3 ( B ) Immunohistochemistry for dystrophin expression in body - wide muscles from mdx mice treated with PMO - M or PMO - M ( + MOTS - c ) ( scale bar : 100 μm ) . TA refers to tibialis anterior . ( C , D ) Western blot ( C ) and quantitative analysis ( D ) for dystrophin expression in body - wide muscles from mdx mice treated with PMO - M or PMO - M ( + MOTS - c ) ( n = 3 ) . 5 µg , 10 µg and 20 µg total protein from C57BL / 6 and 50 µg of muscle samples from untreated and treated mdx mice were loaded . ( E Immunohistochemistry of dystrophin - positive fibres in body - wide muscles from mdx mice treated with PMO at 12 . 5 mg / kg / week for 3 weeks mixed with MOTS - c ( 500 μg ) ( PMO - M ) intravenously ( scale bar : 100 μm ) ( n = 3 ; * p < 0 . 05 ) . PMO represents PMO in saline . F ) quantitative analysis of dystrophin - positive fibres in body - wide muscles from mdx mice treated with PMO at 12 . 5 mg / kg / week for 3 weeks mixed with MOTS - c ( 500 μg ) ( PMO - M ) intravenously ( scale bar : 100 μm ) ( n = 3 ; * p < 0 . 05 ) . PMO represents PMO in saline . ( G , H ) Western blot ( G ) and quantitative analysis ( H ) of dystrophin expression in body - wide muscles from mdx mice treated with PMO at 12 . 5 mg / kg / week for 3 weeks mixed with MOTS - c ( 500 μg ) intravenously ( n = 3 ; * p < 0 . 05 ) . 0 . 5 µg , 1 µg , 2 . 5 µg , 5 µg and 10 µg total protein from C57BL / 6 and 50 µg of muscle samples from untreated and treated mdx mice were loaded . α - actinin was used as the loading control . Fold change refers to PMO - M relative to PMO alone .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3 (B) Immunohistochemistry for dystrophin expression in body-wide muscles from mdx mice treated with PMO-M or PMO-M (+MOTS-c) (scale bar: 100 μm). TA refers to tibialis anterior.  (C, D) Western blot (C) and quantitative analysis (D) for dystrophin expression in body-wide muscles from mdx mice treated with PMO-M or PMO-M (+MOTS-c) (n=3). 5 µg, 10 µg and 20 µg total protein from C57BL/6 and 50 µg of muscle samples from untreated and treated mdx mice were loaded.  (E Immunohistochemistry of dystrophin-positive fibres in body-wide muscles from mdx mice treated with PMO at 12.5 mg/kg/week for 3 weeks mixed with MOTS-c (500 μg) (PMO-M) intravenously (scale bar: 100 μm) (n=3; *p<0.05). PMO represents PMO in saline.  F) quantitative analysis of dystrophin-positive fibres in body-wide muscles from mdx mice treated with PMO at 12.5 mg/kg/week for 3 weeks mixed with MOTS-c (500 μg) (PMO-M) intravenously (scale bar: 100 μm) (n=3; *p<0.05). PMO represents PMO in saline.  (G, H) Western blot (G) and quantitative analysis (H) of dystrophin expression in body-wide muscles from mdx mice treated with PMO at 12.5 mg/kg/week for 3 weeks mixed with MOTS-c (500 μg) intravenously (n=3; *p<0.05). 0.5 µg, 1 µg, 2.5 µg, 5 µg and 10 µg total protein from C57BL/6 and 50 µg of muscle samples from untreated and treated mdx mice were loaded. α-actinin was used as the loading control. Fold change refers to PMO-M relative to PMO alone.


Figure 4 Immunohistochemistry of dystrophin - positive fibres in body - wide muscles from mdx mice treated with repeated doses of PMO - M ( n = 4 ) or PMO alone ( n = 3 ) ( scale bar : 100 μm ) ( * p < 0 . 05 , * * p < 0 . 001 ; two - tailed t test ) . quantitative analysis of dystrophin - positive fibres in body - wide muscles from mdx mice treated with repeated doses of PMO - M ( n = 4 ) or PMO alone ( n = 3 ) ( scale bar : 100 μm ) ( * p < 0 . 05 , * * p < 0 . 001 ; two - tailed t test ) . ( D ) Western blot for dystrophin expression in body - wide muscles from mdx mice treated with PMO - M or PMO alone . 0 . 5 µg , 2 . 5 µg , 5 µg , and 15 µg total protein from C57BL / 6 and 50 µg of muscle samples from untreated and treated mdx mice were loaded . ( E ) Quantitative analysis of dystrophin expression in body - wide muscles from mdx mice treated with PMO - M ( n = 4 ) or PMO alone ( n = 3 ) ( * p < 0 . 05 ; two - tailed t test ) . ( F ) Muscle function was assessed to determine the physical improvement with grip strength test for mdx mice treated with PMO - M ( n = 4 ) or PMO alone ( n = 3 ) , untreated mdx controls ( n = 3 ) and C57BL / 6 ( n = 3 ) ( * p < 0 . 05 , * * p < 0 . 001 , One way - ANOVA post hoc Student - Newman - Keuls test ) . ( G ) Measurement of muscle endurance with the running wheel test for wild - type C57BL / 6 ( C57 ) ( n = 3 ) , untreated mdx controls ( mdx ) ( n = 3 ) and mdx mice treated with PMO - M ( n = 4 ) or PMO alone ( n = 3 ) ( * p < 0 . 05 , One way - ANOVA post hoc Student - Newman - Keuls test ) . Muscle functional tests were conducted two weeks after last injection .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4 Immunohistochemistry of dystrophin-positive fibres in body-wide muscles from mdx mice treated with repeated doses of PMO-M (n=4) or PMO alone (n=3) (scale bar: 100 μm) (*p<0.05, **p<0.001; two-tailed t test).  quantitative analysis of dystrophin-positive fibres in body-wide muscles from mdx mice treated with repeated doses of PMO-M (n=4) or PMO alone (n=3) (scale bar: 100 μm) (*p<0.05, **p<0.001; two-tailed t test).  (D) Western blot for dystrophin expression in body-wide muscles from mdx mice treated with PMO-M or PMO alone. 0.5 µg, 2.5 µg, 5 µg, and 15 µg total protein from C57BL/6 and 50 µg of muscle samples from untreated and treated mdx mice were loaded.  (E) Quantitative analysis of dystrophin expression in body-wide muscles from mdx mice treated with PMO-M (n=4) or PMO alone (n=3) (*p<0.05; two-tailed t test).  (F) Muscle function was assessed to determine the physical improvement with grip strength test for mdx mice treated with PMO-M (n=4) or PMO alone (n=3), untreated mdx controls (n=3) and C57BL/6 (n=3) (*p<0.05, **p<0.001, One way-ANOVA post hoc Student-Newman-Keuls test).  (G) Measurement of muscle endurance with the running wheel test for wild-type C57BL/6 (C57) (n=3), untreated mdx controls (mdx) (n=3) and mdx mice treated with PMO-M (n=4) or PMO alone (n=3) (*p<0.05, One way-ANOVA post hoc Student-Newman-Keuls test). Muscle functional tests were conducted two weeks after last injection.


Figure 5 ( A ) Measurement of serum creatine kinase ( CK ) levels in wild - type C57BL / 6 ( C57 ) ( n = 3 ) , untreated mdx controls ( mdx ) ( n = 3 ) and mdx mice treated with PMO - M ( n = 4 ) or PMO alone ( n = 3 ) ( * p < 0 . 05 , One way - ANOVA post hoc Student - Newman - Keuls test ) . ( B ) Re - localization of DAPC components in treated mdx mice to assess dystrophin function and recovery of normal myoarchitecture ( scale bar : 100 μm ) . The arrowheads point to identical myofibers . C57 means wild - type C57BL / 6 ; mdx represents untreated mdx controls . ( C ) Measurement of body - weight changes of wild - type C57BL / 6 ( C57 ) ( n = 3 ) , untreated mdx controls ( mdx ) ( n = 3 ) and mdx mice treated with PMO - M ( n = 4 ) or PMO alone ( n = 3 ) . ( D ) Quantitative analysis of centrally nucleated fibres ( CNFs ) in triceps and quadriceps of wild - type C57BL / 6 ( C57 ) ( n = 3 ) , untreated mdx controls ( mdx ) ( n = 3 ) and mdx mice treated with repeated doses of PMO - M ( n = 4 ) or PMO ( n = 3 ) ( * p < 0 . 05 , * * p < 0 . 001 , One way - ANOVA post hoc Student - Newman - Keuls test ) . ( E ) Measurement of cross - sectional area ( CSA ) of muscle fibres from mdx mice treated with repeated doses of PMO - M ( n = 4 ) or PMO ( n = 3 ) ( * p < 0 . 05 , two - tailed t test ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5 (A) Measurement of serum creatine kinase (CK) levels in wild-type C57BL/6 (C57) (n=3), untreated mdx controls (mdx) (n=3) and mdx mice treated with PMO-M (n=4) or PMO alone (n=3) (*p<0.05, One way-ANOVA post hoc Student-Newman-Keuls test).  (B) Re-localization of DAPC components in treated mdx mice to assess dystrophin function and recovery of normal myoarchitecture (scale bar: 100 μm). The arrowheads point to identical myofibers. C57 means wild-type C57BL/6; mdx represents untreated mdx controls.  (C) Measurement of body-weight changes of wild-type C57BL/6 (C57) (n=3), untreated mdx controls (mdx) (n=3) and mdx mice treated with PMO-M (n=4) or PMO alone (n=3).  (D) Quantitative analysis of centrally nucleated fibres (CNFs) in triceps and quadriceps of wild-type C57BL/6 (C57) (n=3), untreated mdx controls (mdx) (n=3) and mdx mice treated with repeated doses of PMO-M (n=4) or PMO (n=3) (*p<0.05, **p<0.001, One way-ANOVA post hoc Student-Newman- Keuls test).  (E) Measurement of cross-sectional area (CSA) of muscle fibres from mdx mice treated with repeated doses of PMO-M (n=4) or PMO (n=3) (*p<0.05, two-tailed t test).


Figure 6 ( A Measurement of serum indices including liver enzymes ( AST , ALT and GGT ) from wild - type C57BL / 6 ( C57 ) ( n = 3 ) , untreated mdx controls ( mdx ) ( n = 3 ) and mdx mice treated with PMO - M ( n = 4 ) or PMO alone ( n = 3 ) to reflect liver and kidney functions ( * p < 0 . 05 , One way - ANOVA post hoc Student - Newman - Keuls test ) . B ) Measurement of kidney markers ( CREA and UA ) from wild - type C57BL / 6 ( C57 ) ( n = 3 ) , untreated mdx controls ( mdx ) ( n = 3 ) and mdx mice treated with PMO - M ( n = 4 ) or PMO alone ( n = 3 ) to reflect liver and kidney functions ( * p < 0 . 05 , One way - ANOVA post hoc Student - Newman - Keuls test ) . ( C ) Morphological examination of liver and kidney from wild - type C57BL / 6 ( C57 ) , untreated mdx controls ( mdx ) and treated mdx mice ( scale bar : 100 μm ) . ( D Immunohistochemistry of macrophages , T cells and monocytes in quadriceps and diaphragm from wild - type C57BL / 6 ( C57 ) ( n = 3 ) , untreated mdx controls ( mdx ) ( n = 3 ) and mdx mice treated with PMO - M ( n = 4 ) or PMO ( n = 3 ) ( scale bar : 100 μm ) . The arrowheads point to CD68 + macrophages , CD3 + T cells or CD11b + monocytes ( * p < 0 . 05 , * * p < 0 . 001 , One way - ANOVA post hoc Student - Newman - Keuls test ) . E ) quantification of macrophages , T cells and monocytes in quadriceps and diaphragm from wild - type C57BL / 6 ( C57 ) ( n = 3 ) , untreated mdx controls ( mdx ) ( n = 3 ) and mdx mice treated with PMO - M ( n = 4 ) or PMO ( n = 3 ) ( scale bar : 100 μm ) . The arrowheads point to CD68 + macrophages , CD3 + T cells or CD11b + monocytes ( * p < 0 . 05 , * * p < 0 . 001 , One way - ANOVA post hoc Student - Newman - Keuls test ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6 (A Measurement of serum indices including liver enzymes (AST, ALT and GGT) from wild-type C57BL/6 (C57) (n=3), untreated mdx controls (mdx) (n=3) and mdx mice treated with PMO-M (n=4) or PMO alone (n=3) to reflect liver and kidney functions (*p<0.05, One way-ANOVA post hoc Student-Newman-Keuls test).  B) Measurement of kidney markers (CREA and UA) from wild-type C57BL/6 (C57) (n=3), untreated mdx controls (mdx) (n=3) and mdx mice treated with PMO-M (n=4) or PMO alone (n=3) to reflect liver and kidney functions (*p<0.05, One way-ANOVA post hoc Student-Newman-Keuls test).  (C) Morphological examination of liver and kidney from wild-type C57BL/6 (C57), untreated mdx controls (mdx) and treated mdx mice (scale bar: 100 μm).  (D Immunohistochemistry of macrophages, T cells and monocytes in quadriceps and diaphragm from wild-type C57BL/6 (C57) (n=3), untreated mdx controls (mdx) (n=3) and mdx mice treated with PMO-M (n=4) or PMO (n=3) (scale bar: 100 μm). The arrowheads point to CD68+ macrophages, CD3+ T cells or CD11b+ monocytes (*p<0.05, **p<0.001, One way-ANOVA post hoc Student-Newman-Keuls test).  E) quantification of macrophages, T cells and monocytes in quadriceps and diaphragm from wild-type C57BL/6 (C57) (n=3), untreated mdx controls (mdx) (n=3) and mdx mice treated with PMO-M (n=4) or PMO (n=3) (scale bar: 100 μm). The arrowheads point to CD68+ macrophages, CD3+ T cells or CD11b+ monocytes (*p<0.05, **p<0.001, One way-ANOVA post hoc Student-Newman-Keuls test).


Figure 1Plot showing the average Z - scores of 210 endolysosomal genes plotted against the negative log10 of the P values from the one - sample t - test of whether average Z - score ≠ 0 . Green circles represent significantly upregulated genes ( Z > 0 and P < 0 . 05 ) . Magenta circles represent significantly downregulated genes ( Z < 0 and P < 0 . 05 ) . Grey circles represent genes whose expression is not significantly changed ( P > 0 . 05 ) . All comparisons were between cancers and normal cells from the same tissue . Dashed line shows position on Y - axis that corresponds to P = 0 . 05 . Positions of MCOLN1 ( red circle ) , VAC14 ( blue circle ) , and MTM1 ( yellow circle ) are indicated . Inset , schematic showing that Mtm1 and Vac14 regulate the levels of PI ( 3 , 5 ) P2 , and thereby , influence TRPML1 activity . Unsupervised hierarchical clustering of Pearson ' s coefficients of pairwise correlation of gene expression reveal 4 indicated clusters . Violin plots of average Z - scores of genes that belong to the four clusters derived from hierarchical clustering . The thick horizontal dashed lines in each sample set represent median ; thin dashed lines represent the quartiles ; polygons represent density estimates of data and extend to extreme values . Statistical tests employed were 1 - sample t - test or Wilcoxon signed rank test to examine significance of differences from a hypothetical value of 0 . GSEA conducted on endolysosomal genes ranked on the basis of increasing Z - scores . Positions of the CLEAR target genes are indicated . Bar graph showing the relative expression of the indicated genes in the indicated cell types . Values were normalized to respective ' HN31 + HRAS shRNA ' average and represent mean ± SEM . Data points represent values from biological replicates . Statistical test employed was Student ' s t - test . Western blots performed on extracts isolated from the indicated cells probed with antibodies against Tfeb and tubulin . Molecular weights are shown on the left . Bar graph shows relative Tfeb levels in the indicated samples . Values were normalized to ' HN31 + HRAS shRNA ' and represent mean ± SEM . Data points represent values from biological replicates . Statistical test employed was Student ' s t - test . Bar graph showing the relative expression of the indicated genes in the indicated cell types . Values were normalized to respective ' HN31 + HRAS shRNA ' average and represent mean ± SEM . Data points represent values from biological replicates . Statistical test employed was Student ' s t - test . Bar graph showing the relative expression of MCOLN1 in the indicated cell types . Values were normalized to HT1197 average and represent mean ± SEM . Data points represent values from biological replicates . Statistical test employed was Student ' s t - test .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1Plot showing the average Z-scores of 210 endolysosomal genes plotted against the negative log10 of the P values from the one-sample t-test of whether average Z-score ≠ 0. Green circles represent significantly upregulated genes (Z > 0 and P < 0.05). Magenta circles represent significantly downregulated genes (Z < 0 and P < 0.05). Grey circles represent genes whose expression is not significantly changed (P > 0.05). All comparisons were between cancers and normal cells from the same tissue. Dashed line shows position on Y-axis that corresponds to P = 0.05. Positions of MCOLN1 (red circle), VAC14 (blue circle), and MTM1 (yellow circle) are indicated. Inset, schematic showing that Mtm1 and Vac14 regulate the levels of PI(3,5)P2, and thereby, influence TRPML1 activity.Unsupervised hierarchical clustering of Pearson's coefficients of pairwise correlation of gene expression reveal 4 indicated clusters. Violin plots of average Z-scores of genes that belong to the four clusters derived from hierarchical clustering. The thick horizontal dashed lines in each sample set represent median; thin dashed lines represent the quartiles; polygons represent density estimates of data and extend to extreme values. Statistical tests employed were 1-sample t-test or Wilcoxon signed rank test to examine significance of differences from a hypothetical value of 0.GSEA conducted on endolysosomal genes ranked on the basis of increasing Z-scores. Positions of the CLEAR target genes are indicated.Bar graph showing the relative expression of the indicated genes in the indicated cell types. Values were normalized to respective 'HN31+HRAS shRNA' average and represent mean±SEM. Data points represent values from biological replicates. Statistical test employed was Student's t-test.Western blots performed on extracts isolated from the indicated cells probed with antibodies against Tfeb and tubulin. Molecular weights are shown on the left. Bar graph shows relative Tfeb levels in the indicated samples. Values were normalized to 'HN31+HRAS shRNA' and represent mean±SEM. Data points represent values from biological replicates. Statistical test employed was Student's t-test.Bar graph showing the relative expression of the indicated genes in the indicated cell types. Values were normalized to respective 'HN31+HRAS shRNA' average and represent mean±SEM. Data points represent values from biological replicates. Statistical test employed was Student's t-test.Bar graph showing the relative expression of MCOLN1 in the indicated cell types. Values were normalized to HT1197 average and represent mean±SEM. Data points represent values from biological replicates. Statistical test employed was Student's t-test.


Figure 2Graphs showing the changes in the fura - 2 ratio in cells of the indicated genotypes in response to ML - SA1 and GPN . Arrows serve as event markers that denote the points of time at which the drugs were added . Grey lines on top represent the time at which the bath Ca2 + was removed . All values represent mean ± SEM from 3 biological replicates . Graphs showing the changes in the fura - 2 ratio in cells of the indicated genotypes in response to ML - SA1 except that the baseline ratios for both genotypes were adjusted to 0 at the start of the experiment . Lines on the right indicate peak amplitudes of Ca2 + release in response to the application of ML - SA1 . All values represent mean ± SEM from 3 biological replicates . Graphs showing the changes in the fura - 2 ratio in cells of the indicated genotypes in response to GPN . the baseline ratios for both genotypes were adjusted to 0 at the start of the experiment . Lines on the right indicate peak amplitudes of Ca2 + release Ca2 + release was evoked using GPN . Peak amplitudes of Ca2 + release in cells of the indicated genotypes in response to ML - SA1 ( E ) All values represent mean ± SEM . Data points represent values from biological replicates . Statistical test employed was Student ' s t - test . Peak amplitudes of Ca2 + release in cells of the indicated genotypes in response to GPN ( F ) . All values represent mean ± SEM . Data points represent values from biological replicates . Statistical test employed was Student ' s t - test . Ratio of the amplitudes of Ca2 + release in cells of the indicated genotypes in response to ML - SA1 and GPN . All values represent mean ± SEM . Data points represent values from biological replicates . Statistical test employed was Student ' s t - test . Total Ca2 + release in cells of the indicated genotypes in response to ML - SA1 and GPN . All values represent mean ± SEM . Data points represent values from biological replicates . Statistical test employed was Student ' s t - test .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2Graphs showing the changes in the fura-2 ratio in cells of the indicated genotypes in response to ML-SA1 and GPN. Arrows serve as event markers that denote the points of time at which the drugs were added. Grey lines on top represent the time at which the bath Ca2+ was removed. All values represent mean±SEM from 3 biological replicates.Graphs showing the changes in the fura-2 ratio in cells of the indicated genotypes in response to ML-SA1 except that the baseline ratios for both genotypes were adjusted to 0 at the start of the experiment. Lines on the right indicate peak amplitudes of Ca2+ release in response to the application of ML-SA1. All values represent mean±SEM from 3 biological replicates.Graphs showing the changes in the fura-2 ratio in cells of the indicated genotypes in response to GPN. the baseline ratios for both genotypes were adjusted to 0 at the start of the experiment. Lines on the right indicate peak amplitudes of Ca2+ release Ca2+ release was evoked using GPN.Peak amplitudes of Ca2+ release in cells of the indicated genotypes in response to ML-SA1 (E) All values represent mean±SEM. Data points represent values from biological replicates. Statistical test employed was Student's t-test.Peak amplitudes of Ca2+ release in cells of the indicated genotypes in response to GPN (F). All values represent mean±SEM. Data points represent values from biological replicates. Statistical test employed was Student's t-test.Ratio of the amplitudes of Ca2+ release in cells of the indicated genotypes in response to ML-SA1 and GPN. All values represent mean±SEM. Data points represent values from biological replicates. Statistical test employed was Student's t-test.Total Ca2+ release in cells of the indicated genotypes in response to ML-SA1 and GPN. All values represent mean±SEM. Data points represent values from biological replicates. Statistical test employed was Student's t-test.