Brain sAPPβ shows an - related increase in APP23 , APP24 , APP51 micesAPPβ was measured in Triton X - 100 brain extracts from largely same mice as analyzed in Figs1 and2 is expressed as percentages of measured in youngest group . Swedish sAPPβ showed an - dependent increase in APP23 mice following a linear trend ( F ( 1 , 83 ) = 52 . 914 , P < 0 . 001 ) ; APP23 from two independent batches were included in this ( see Materials Methods Supplementary Fig S2 for details ) . Swedish sAPPβ showed an - dependent increase in APP24mice following a linear trend ( F ( 1 , 84 ) = 11 . 264 , P = 0 . 001 ) . Human wild - type sAPPβ showed an - dependent increase in APP51 following a quadratic trend ( F ( 1 , 18 ) = 68 . 980 , P < 0 . 001 ) . Data information : Post hoc Dunnett ' s group comparisons were always conducted between youngest group other groups . data are represented as group means ± ; * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 . For absolute values , see Supplementary Fig S2 .

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Brain sAPPβ shows an age-related increase in APP23, APP24, and APP51 micesAPPβ was measured in Triton X-100 brain extracts from largely the same mice as analyzed in Figs1 and2 and is expressed as percentages of levels measured in the youngest age group.Swedish sAPPβ showed an age-dependent increase in APP23 mice following a linear trend (F(1, 83) = 52.914, P < 0.001); APP23 from two independent batches were included in this analysis (see Materials and Methods and Supplementary Fig S2 for details).Swedish sAPPβ showed an age-dependent increase in APP24mice following a linear trend (F(1, 84) = 11.264, P = 0.001).Human wild-type sAPPβ showed an age-dependent increase in APP51 following a quadratic trend (F(1, 18) = 68.980, P < 0.001).Data information: Post hoc Dunnett's test group comparisons were always conducted between the youngest group and all other groups. All data are represented as group means ± SEM; *P < 0.05; **P < 0.01; and ***P < 0.001. For absolute values, see Supplementary Fig S2.


( B ) of that expressing ZGA reporter ( MERVL : : tdTomato ) after injection with a siRNA - repressor or mRNA - inducer . N , total number of analyzed for each condition . Error bars , mean ± S . D . ; n = 3 biological replicates per group .

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(B) Quantification of embryos that expressing the ZGA reporter (MERVL::tdTomato) after injection with a siRNA-repressor or mRNA-inducer. N, the total number of embryos analyzed for each condition. Error bars, mean ± S.D.; n = 3 biological replicates per group.


( C ) Representative image of SCNT derived by different methods . SCNT produced by transfer of MERVL : : tdTomato cumulus ( B6D2F1 background ) into WT enucleated oocytes . n = 3 biological replicates per group . Scale bar , 100 μm .

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(C) Representative fluorescence image of SCNT embryos derived by different methods. The SCNT embryos produced by transfer of MERVL::tdTomato cumulus cells (B6D2F1 background) into WT enucleated oocytes. n = 3 biological replicates per group. Scale bar, 100 μm.


( D ) Stacked bar plots show fraction of at blastocyst stages after injection with different mRNA , as indicated . Error bars , mean ± S . D . ; n = 3 biological replicates per group ; N , total number of analyzed for each condition ; * * P < 0 . 01 , * * * P < 0 . 001 as compared with control group , by two - Student ' s t - .

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(D) Stacked bar plots show the fraction of embryos at blastocyst stages after injection with different mRNA, as indicated. Error bars, mean ± S.D.; n = 3 biological replicates per group; N, total number of embryos analyzed for each condition; **P < 0.01, ***P < 0.001 as compared with control group, by two-tailed Student's
 t-test.


( E ) Representative live - of dynamic ZGA - reporter expression during SCNT development . time after initial observation is shown at bottom of image . n = 3 biological replicates per group . Scale bar , 50 μm .

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(E) Representative live-cell images of dynamic ZGA-reporter expression during SCNT embryo development. The time after initial observation is shown at the bottom of the image. n = 3 biological replicates per group. Scale bar, 50 μm.


( G ) Preimplantation development of SCNT . of reaching each indicated stage is shown . D - SCNT produced by transfer of dox - Dux cumulus ( B6D2F1 background ) into WT enucleated oocytes ( B6D2F1 background ) . Error bars , mean ± S . D . ; n = 3 biological replicates per group . Scale bar , 100 μm .

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(G) Preimplantation development of SCNT embryos. The percentage of embryos reaching each indicated stage is shown. The D-SCNT embryos produced by transfer of dox-Dux cumulus cell (B6D2F1 background) into WT enucleated oocytes (B6D2F1 background). Error bars, mean ± S.D.; n = 3 biological replicates per group. Scale bar, 100 μm.


( J ) Image of full - term cloned mice derived by canonical SCNT D - SCNT method ( left ) . birth rate of SCNT derived by different methods is indicated ( right ) . N , total number of cloned pups obtained from each condition .

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(J) Image of full-term cloned mice derived by canonical SCNT and D-SCNT method (left). The birth rate of SCNT embryos derived by different methods is indicated (right). N, the total number of cloned pups obtained from each condition.


( B ) Durations of dox addition at different time points during chemical induction ( upper ) assessment of AP + colonies ( bottom ) . Error bars , mean ± S . D . ; n = 3 biological replicates per condition ; * * P < 0 . 01 , * * * P < 0 . 001 as compared with condition - 1 , by two - Student ' s t - . n . s . , not significant .

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(B) Durations of dox addition at different time points during chemical induction (upper) and assessment of AP+ colonies (bottom). Error bars, mean ± S.D.; n = 3 biological replicates per condition; **P < 0.01, ***P < 0.001 as compared with condition-1, by two-tailed Student's
 t-test. n.s., not significant.


( C ) Morphological changes at distinct time points during D - CiPS induction . Scale bar , 100 μm .

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(C) Morphological changes at distinct time points during D-CiPS induction. Scale bar, 100 μm.


( D ) Representative of number of AP + positive colonies . n = 5 biological replicates per condition .

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(D) Representative images of the number of AP+ positive colonies. n = 5 biological replicates per condition.


( E ) RT - qPCR of select ZGA - related genes in dox - untreated D - CiPSCs canonical CiPSCs at passage 1 . value in CiPSCs was set as 1 , data shown are mean expression values relative to GAPDH . Error bars , mean ± S . E . M . ; n = 3 biological replicates per group ; * * * P < 0 . 001 by two - Student ' s t - .

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(E) RT-qPCR analysis of select ZGA-related genes in dox-untreated D-CiPSCs and canonical CiPSCs at passage 1. The value in CiPSCs was set as 1, and data shown are mean expression values relative to GAPDH. Error bars, mean ± S.E.M.; n = 3 biological replicates per group; ***P < 0.001 by two-tailed Student's t-test.


( F ) Representative immunostaining of D - CiPSCs expressing pluripotency markers . n = 5 biological replicates . Scale bar , 50 μm .

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(F) Representative immunostaining images of D-CiPSCs expressing pluripotency markers. n = 5 biological replicates. Scale bar, 50 μm.


( G ) Histology by H & E of teratoma tissues derived by D - CiPS . n = 3 biological replicates . Scale bar , 200 μm .

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(G) Histology by H&E staining of teratoma tissues derived by D-CiPS cells. n = 3 biological replicates. Scale bar, 200 μm.


( I ) Karyotype of D - CiPSCs canonical CiPSCs . majority of D - CiPSCs CiPSCs possessed a normal karyotype . Scale bar , 10 μm .

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(I) Karyotype analysis of D-CiPSCs and canonical CiPSCs. The majority of D-CiPSCs and CiPSCs cells possessed a normal karyotype. Scale bar, 10 μm.


( J ) Bisulfite sequencing of demethylation of Oct4 Nanog promoters in D - CiPSCs or canonical CiPSCs . Filled empty squares represent methylated unmethylated CpGs , respectively .

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(J) Bisulfite sequencing analysis of demethylation of Oct4 and Nanog promoters in D-CiPSCs or canonical CiPSCs. Filled and empty squares represent methylated and unmethylated CpGs, respectively.


Ookinete formation in vitro . After 12 h of culture , ookinetes were Giemsa - stained analyzed for ookinete . ookinete conversion rate was calculated as number of mature ookinetes per 100 gametocytes .

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Ookinete formation in vitro. After 12 h of culture, the ookinetes were Giemsa-stained and analyzed for ookinete morphology. The ookinete conversion rate was calculated as the number of mature ookinetes per 100 female gametocytes.


Oocyst in mosquitoes at day 7 post blood - feeding . x / y on top is number of mosquitoes containing oocyst / number of mosquitoes dissected ; number is mosquito infection prevalence .

Oocyst counts in the mosquitoes at day 7 post blood-feeding. x/y on the top is the number of mosquitoes containing oocyst/the number of mosquitoes dissected; the percentage number is the mosquito infection prevalence.


Co - of P28 GAP45 ( IMC ) in gametes ( P28 + only ) zygotes ( P28 + / GAP45 + ) of 17XNL , Δap2 - o3 Δcdpk4 strains . Percentages of gametes zygotes were indicated respectively . n is number of counted . Scale bars = 5 μm . Note : signal of P28 GAP45 is reduced similarly in Δap2 - o3 gametes zygotes compared to that of 17XNL .

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Co-staining of P28 and GAP45 (IMC protein) in female gametes (P28+ only) and zygotes (P28+/GAP45+) of the 17XNL, Δap2-o3 and Δcdpk4 strains. Percentages of female gametes and zygotes were indicated respectively. n is the number of cells counted. Scale bars = 5 μm. Note: the signal of P28 and GAP45 is reduced similarly in all Δap2-o3 female gametes and zygotes compared to that of 17XNL.


Day 7 midgut oocyst from mosquitoes infected with , including 17XNL , Δap2 - o3 , Δmap2 , or Δnek4 alone as well as mixtures of Δmap2 / Δnek4 , Δap2 - o3 / Δmap2 , Δap2 - o3 / Δnek4 . Δnek4 Δmap2 are gamete - defect , respectively . n is number of mosquitoes dissected , Mann - Whitney applied , two experiments repeated .

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Day 7 midgut oocyst counts from mosquitoes infected with parasites, including 17XNL, Δap2-o3, Δmap2, or Δnek4 strain alone as well as mixtures of Δmap2/Δnek4, Δap2-o3/Δmap2, and Δap2-o3/Δnek4. Δnek4 and Δmap2 are female and male gamete-defect parasites, respectively. n is the number of mosquitoes dissected, Mann-Whitney test applied, two experiments repeated.


Transcript expression by - seq of 26 genes involving replication repair between DFsc7 ( WT ) DFsc7 ; Δap2 - o3 ( KO ) strains .

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Transcript expression heatmap by RNA-seq of 26 genes involving DNA replication and DNA repair between DFsc7 (WT) and DFsc7;Δap2-o3 (KO) strains.


Representative microscopy of mCherry DPOD2 : : GFP expression in gametocytes of DTS1 ( ccp2 : : mCherry ; dpod2 : : gfp ) DTS1 ; Δap2 - o3 strains . mCherry is specifically expressed in gametocytes . Flow cytometry detection of mCherry DPOD2 : : GFP expression in gametocytes of DTS1 DTS1 ; Δap2 - o3 strains . gametocyte populations are circled by solid dashed respectively . E , F Similar ( as in C , D ) in DTS2 ( ccp2 : : mCherry ; dpod1 : : gfp ) DTS2 ; Δap2 - o3 strains . G , H Similar ( as in C , D ) in DTS3 ( ccp2 : : mCherry ; rpa1 : : gfp ) DTS3 ; Δap2 - o3 strains . I , J Similar ( as in C , D ) in DTS4 ( ccp2 : : mCherry ; mcm7 : : gfp ) DTS4 ; Δap2 - o3 strains .

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Representative fluorescence microscopy images of mCherry and DPOD2::GFP expression in female and male gametocytes of the DTS1 (ccp2::mCherry;dpod2::gfp) and DTS1;Δap2-o3 strains. mCherry is specifically expressed in female gametocytes. Flow cytometry detection of mCherry and DPOD2::GFP expression in female and male gametocytes of the DTS1 and DTS1;Δap2-o3 strains. The female and male gametocyte populations are circled by solid and dashed line respectively. E,F Similar analysis (as in C, D) in DTS2 (ccp2::mCherry;dpod1::gfp) and DTS2;Δap2-o3 strains. G,H Similar analysis (as in C, D) in DTS3 (ccp2::mCherry;rpa1::gfp) and DTS3;Δap2-o3 strains. I,J Similar analysis (as in C, D) in DTS4 (ccp2::mCherry;mcm7::gfp) and DTS4;Δap2-o3 strains.


EMSA using recombinant GST - fused AP2 domain of AP2 - O3 a synthesized probe containing three repeats of predicted motif . GST was used as a negative control . An arrowhead indicates shifted band .

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EMSA using the recombinant GST-fused AP2 domain of AP2-O3 and a synthesized DNA probe containing three repeats of predicted motif. GST was used as a negative control. An arrowhead indicates the shifted band.


ChIP - qPCR validation of binding between AP2 - O3 upstream region of four genes regions detected via ChIP - qPCR are indicated by pink ( in peak ) purple ( out of peak ) . mean ± from three independent experiments .

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ChIP-qPCR validation of the binding between AP2-O3 and the upstream region of four male genes The regions detected via ChIP-qPCR are indicated by pink line (in the peak) and purple line (out of the peak). mean±SEM from three independent experiments.


qRT - PCR detecting mRNA transcript level of ten selected highly expressed genes numbers indicate gene ID .

qRT-PCR detecting mRNA transcript level of ten selected highly expressed genes The numbers indicate the gene ID.


Western blot of GAP45 expression in non - activated ( NAG ) activated gametocyte ( AG ) of 17XNL Δap2 - o3 strains . BiP as loading control .

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Western blot of GAP45 protein expression in non-activated (NAG) and activated gametocyte (AG) of 17XNL and Δap2-o3 strains. BiP as loading control.


IFA of AP2 - O3 expression in activated gametocytes of Pccp2 strains .

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IFA of AP2-O3 protein expression in activated gametocytes of Pccp2 strains.


( a ) Brains of ' rescued ' mice ( Becn1 − / − ; Becn1EGFP / + ) of control Becn1 + / + were lysed subjected to immune isolation using an anti - GFP antibody . bound to anti - GFP antibody were isolated , detected identified by SDS - PAGE , Coomassie brilliant blue mass spectrometry . Seven bands pulled down by anti - GFP antibody specifically from ' rescued ' mice lysate are numbered ( this is a copy of image from our previous report with modification ) .

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(a) Brains of the 'rescued' mice (Becn1−/−; Becn1EGFP/+) and of control Becn1+/+ littermates were lysed and subjected to immune isolation using an anti-GFP antibody. Proteins bound to anti-GFP antibody were isolated, detected and identified by SDS-PAGE, Coomassie brilliant blue staining and mass spectrometry. Seven bands pulled down by anti-GFP antibody specifically from 'rescued' mice lysate are numbered (this is a copy of image from our previous report with modification).


( b ) Mouse brain lysates were subjected to co - immunoprecipitation ( co - IP ) followed by WB using indicated antibodies .

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(b) Mouse brain lysates were subjected to co-immunoprecipitation (co-IP) followed by WB analysis using the indicated antibodies.


( c ) Mouse brain lysates were subjected to co - IP by NRBF2 antibody followed by WB using indicated antibodies .

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(c) Mouse brain lysates were subjected to co-IP by NRBF2 antibody and followed by WB analysis using the indicated antibodies.


( d ) Gel filtration . supernatant fraction of mouse brain lysates was subjected to a Superdex 200 HR 10 / 30 column each fraction was immunoblotted with indicated antibodies . Relative amounts of each fraction determined by Odyssey software are plotted .

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(d) Gel filtration analysis. The supernatant fraction of mouse brain lysates was subjected to a Superdex 200 HR 10/30 column and each fraction was immunoblotted with the indicated antibodies. Relative amounts of each fraction determined by Odyssey software are plotted.


( e ) deficiency of Atg14L impairs interaction between Beclin 1 NRBF2 . NIH3T3 were transfected with Atg14L or NRBF2 RNAi for 72 h , respectively , then were lysed subjected to IP using Beclin 1 antibody . immunoprecipitants were immunoblotted using indicated antibodies .

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(e) The deficiency of Atg14L impairs the protein interaction between Beclin 1 and NRBF2. NIH3T3 cells were transfected with Atg14L or NRBF2 RNAi for 72 h, respectively, and then were lysed and subjected to IP using Beclin 1 antibody. The immunoprecipitants were immunoblotted using the indicated antibodies.


( f ) Direct binding between Atg14L NRBF2 . Flag - tagged Atg14L purified from HEK293T overexpressing Flag - Atg14L was incubated with GST or NRBF2 - GST purified from Escherichia coli . GST pull - down was performed to examine direct binding between Flag - Atg14L NRBF2 - GST .

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(f) Direct binding between Atg14L and NRBF2. Flag-tagged Atg14L purified from HEK293T cells overexpressing Flag-Atg14L was incubated with GST or NRBF2-GST purified from Escherichia coli. The GST pull-down assay was performed to examine the direct binding between Flag-Atg14L and NRBF2-GST.


( b ) NRBF2 KO MEFs were transfected with CFP , NRBF2 - CFP , dMIT - CFP or dCCD - CFP for 48 h . Atg14L - linked Vps34 was pulled down by an anti - Atg14L antibody subjected to KA . IP products inputs were immunoblotted using antibodies indicated . ( c ) of IPed Vps34 kinase activity ( PI ( 3 ) P ) , Vps34 Vps15 versus IPed Atg14L . ( Data are shown as mean ± s . e . m . , P values are indicated on figures , multiple t - are followed by Bonferroni correction , n = 4 ( otherwise indicated , n means number of independent experiments ; NS , not significant ) ) .

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(b) NRBF2 KO MEFs were transfected with CFP, NRBF2-CFP, dMIT-CFP or dCCD-CFP for 48 h. Atg14L-linked Vps34 was pulled down by an anti-Atg14L antibody and subjected to KA. IP products and inputs were immunoblotted using the antibodies indicated. (c) Quantification of IPed Vps34 kinase activity (PI(3)P), Vps34 and Vps15 protein versus IPed Atg14L protein. (Data are shown as mean±s.e.m., P values are indicated on the figures, multiple t-test are followed by Bonferroni correction, n=4 (otherwise indicated, n means number of independent experiments; NS, not significant)).


( d ) Overexpression of NRBF2 enhances Atg14L - linked Vps34kinase activity . HEK293T were co - transfected with Myc - Vps34 - Vps15 - His Flag - Atg14L in presence of CFP or NRBF2 - CFP . Atg14L - linked Vps34 is pulled down by an anti - Flag antibody subjected to KA ( upper panel ) . IP products inputs were immunoblotted using antibodies indicated ( upper panel ) . ( e ) of Vps34kinase activity ( lower panel ) shows significant difference between two groups . ( Data are shown as mean ± s . e . m . , P value is indicated on figures , one sample t - versus hypothetical mean 1 , n = 5 ) .

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(d) Overexpression of NRBF2 enhances Atg14L-linked Vps34kinase activity. HEK293T cells were co-transfected with Myc-Vps34-Vps15-His and Flag-Atg14L in the presence of CFP or NRBF2-CFP. Atg14L-linked Vps34 is pulled down by an anti-Flag antibody and subjected to KA (upper panel). IP products and inputs were immunoblotted using the antibodies indicated (upper panel). (e) Quantification of Vps34kinase activity (lower panel) shows the significant difference between two groups. (Data are shown as mean±s.e.m., P value is indicated on the figures, one sample t-test versus hypothetical mean 1, n=5).


( a ) Starvation - induced long - lived degradation is impaired after NRBF2 knockdown . Compared with control ( con ) siRNA , NRBF2 siRNA decreases long - lived degradation ( proteolysis rate ) in NIH3T3 under starvation ( ST ) condition . This difference is diminished when starved are co - treated with wortmannin ( WM , 100 nM ) . ( Data are shown as mean ± s . e . m . , P values are indicated on figures , one - way of variance with Dunnett as post hoc , n = 4 ; NS , not significant ) .

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(a) Starvation-induced long-lived protein degradation is impaired after NRBF2 knockdown. Compared with control (con) siRNA, NRBF2 siRNA decreases long-lived protein degradation (proteolysis rate) in NIH3T3 cells under starvation (ST) condition. This difference is diminished when the starved cells are co-treated with wortmannin (WM, 100 nM). (Data are shown as mean±s.e.m., P values are indicated on the figures, one-way analysis of variance with Dunnett as post hoc test, n=4; NS, not significant).


( b ) Rapamycin ( Rap ) - induced autophagy is impaired in NRBF2 KO . MEFs were treated with ( dimethyl sulfoxide ) DMSO or 50 μg ml − 1 of Rap for 4 h then lysates were immunoprobed with anti - LC3 anti - p62 antibodies . ( c ) LC3 - II p62 were quantified normalized with β - actin . results show that Rap - induced LC3 lipidation ( upper ) p62 degradation ( lower ) were markedly impaired in NRBF2 KO MEFs . ( Data are shown as mean ± s . e . m . , P values are indicated on figures , one sample t - ( WT con versus KO con ) or unpaired t - ( WT Rap versus KO Rap ) ) . p62 blots , n = 6 ; LC3 blots , n = 3 ) .

O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O

(b) Rapamycin (Rap)-induced autophagy is impaired in the NRBF2 KO cells. The MEFs were treated with (dimethyl sulfoxide) DMSO or 50 μg ml−1 of Rap for 4 h then the cell lysates were immunoprobed with anti-LC3 and anti-p62 antibodies. (c) LC3-II and p62 levels were quantified and normalized with β-actin. Quantification results show that Rap-induced LC3 lipidation (upper) and p62 degradation (lower) were markedly impaired in the NRBF2 KO MEFs. (Data are shown as mean±s.e.m., P values are indicated on the figures, one sample t-test (WT con versus KO con) or unpaired t-test (WT Rap versus KO Rap)). p62 blots quantification, n=6; LC3 blots quantification, n=3).


( d , e ) MEFs were subjected to Rap ( 50 μg ml − 1 ) or starvation ( HBSS ) treatment then stained with anti - LC3 ( d ) or anti - WIPI2 ( e ) antibody . ( f ) LC3 WIPI2 per were counted quantified . ( Data are shown as mean ± s . e . m . , P values are indicated on figures , independent samples Kruskal - Wallis followed by pairwise comparisons , n = 30 randomly selected , a representative result from three independent repeats ) .

O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(d,e) The MEFs were subjected to Rap (50 μg ml−1) or starvation (HBSS) treatment then stained with anti-LC3 (d) or anti-WIPI2 (e) antibody. (f) The LC3 and WIPI2 puncta per cell were counted and quantified. (Data are shown as mean±s.e.m., P values are indicated on the figures, independent samples Kruskal-Wallis test followed by pairwise comparisons, n=30 randomly selected cells, a representative result from three independent repeats).


( g ) NRBF2 - CFP stable were starved in HBSS for 4 h then subjected to WIPI2 . Under starved condition , NRBF2 - CFP forms structures that co - localized with WIPI2 . This is a representative image from three independent experiments .

O O O B-MEASURED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O

(g) NRBF2-CFP stable cells were starved in HBSS for 4 h and then subjected to WIPI2 staining. Under starved condition, NRBF2-CFP forms puncta structures that co-localized with WIPI2. This is a representative image from three independent experiments.


( h ) NRBF2 KO MEFs were transfected with CFP , NRBF2 - CFP , dMIT - CFP or dCCD - CFP for 24 h then followed by co - immunostaining with GFP WIPI2 antibodies . ( i ) WIPI2 number per in GFP - positive were counted quantified . ( Data are shown as mean ± s . e . m . , P values are indicated on figures , independent samples Kruskal - Wallis followed by pairwise comparisons , n = 30 randomly selected , a representative result from two independent repeats ) . Scale bars , 20 μm , or indicated on figure . CM , culture medium .

O O O B-CONTROLLED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(h) The NRBF2 KO MEFs were transfected with CFP, NRBF2-CFP, dMIT-CFP or dCCD-CFP for 24 h and then followed by co-immunostaining with GFP and WIPI2 antibodies. (i) The WIPI2 puncta number per cell in GFP-positive cells were counted and quantified. (Data are shown as mean±s.e.m., P values are indicated on the figures, independent samples Kruskal-Wallis test followed by pairwise comparisons, n=30 randomly selected cells, a representative result from two independent repeats). Scale bars, 20 μm, or length indicated on the figure. CM, culture medium.


( a ) NRBF2 KO MEFs display impaired autophagosome acidification . WT NRBF2 KO MEFs are transiently transfected with mCherry - GFP - LC3B . WT MEFs expressing mCherry - GFP - LC3B contain many red - only along with yellow ( presence of both red green ) . number of both red - only yellow markedly increases after rapamycin ( Rap ; 50 μg ml − 1 , 4 h ) or starvation ( HBSS , 4 h ) treatment , suggesting increased autophagolysosomes nascent autophagosomes . In contrast , NRBF2 KO MEFs expressing mCherry - GFP - LC3B contain quite fewer red - only yellow , even after Rap or starvation treatment . ( b ) results show that both numbers of red - only of red - only ( verses total ) in KO MEFs were markedly reduced . ( Data are shown as mean ± s . e . m . , P values are indicated on figures , Mann - Whitney U - , n = 20 randomly selected , a representative result from three independent repeats ) , indicating both autophagolysome number autophagosome acidification rate are reduced in KO . Scale bars , 20 μm or 10 μm ( amplified regions ) .

O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O O 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-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(a) NRBF2 KO MEFs display impaired autophagosome acidification. WT and NRBF2 KO MEFs are transiently transfected with mCherry-GFP-LC3B. WT MEFs expressing mCherry-GFP-LC3B contain many red-only puncta along with yellow (presence of both red and green) puncta. The number of both red-only puncta and yellow puncta markedly increases after rapamycin (Rap; 50 μg ml−1, 4 h) or starvation (HBSS, 4 h) treatment, suggesting the increased autophagolysosomes and nascent autophagosomes. In contrast, NRBF2 KO MEFs expressing mCherry-GFP-LC3B contain quite fewer red-only puncta and yellow puncta, even after Rap or starvation treatment. (b) The quantification results show that both the numbers of red-only puncta and percentage of red-only puncta (verses total puncta) in KO MEFs were markedly reduced. (Data are shown as mean±s.e.m., P values are indicated on the figures, Mann-Whitney U-test, n=20 randomly selected cells, a representative result from three independent repeats), indicating both the autophagolysome number and autophagosome acidification rate are reduced in KO cells. Scale bars, 20 μm or 10 μm (amplified regions).


( c ) Autophagolysome formation is impaired in NRBF2 KO MEFs . WT NRBF2 KO MEFs were transiently transfected with RFP - LC3 then stained with LAMP2 antibody . co - localization between RFP - LC3 LAMP2 are illustrated by profile . RFP - LC3 recruit much stronger LAMP2 signal in WT MEFs than in NRBF2 KO MEFs ( as indicated by arrows ) , suggesting impairment of autophagolysosome formation in KO . Scale bars , 20 μm or 10 μm ( amplified regions ) . This is a representative image from three independent experiments .

O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(c) Autophagolysome formation is impaired in NRBF2 KO MEFs. WT and NRBF2 KO MEFs were transiently transfected with RFP-LC3 then stained with LAMP2 antibody. The co-localization between RFP-LC3 and LAMP2 are illustrated by line profile. RFP-LC3 puncta recruit much stronger LAMP2 signal in WT MEFs than in NRBF2 KO MEFs (as indicated by white arrows), suggesting the impairment of autophagolysosome formation in KO cells. Scale bars, 20 μm or 10 μm (amplified regions). This is a representative image from three independent experiments.


( d ) Overexpression of NRBF2 enhances UVRAG - linked Vps34 kinase activity . HEK293T were co - transfected with Myc - Vps34 - Vps15 - His FLAG - UVRAG , in presence of CFP or NRBF2 - CFP . UVRAG - linked Vps34 was pulled down by an anti - FLAG antibody subjected to KA . IP products inputs are immunoblotted using antibodies indicated . ( e ) of Vps34 kinase activity normalized with immunoprecipitated Vps34 shows significant difference between two groups . ( Data are shown as mean ± s . e . m . , P value is indicated on figures , one sample t - versus hypothetical mean 1 , n = 5 ) .

O O O O O B-CONTROLLED_VAR O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O B-MEASURED_VAR O O 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-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(d) Overexpression of NRBF2 enhances UVRAG-linked Vps34 kinase activity. HEK293T cells were co-transfected with Myc-Vps34-Vps15-His and FLAG-UVRAG, in the presence of CFP or NRBF2-CFP. UVRAG-linked Vps34 was pulled down by an anti-FLAG antibody and subjected to KA. IP products and inputs are immunoblotted using the antibodies indicated. (e) Quantification of Vps34 kinase activity normalized with immunoprecipitated Vps34 shows the significant difference between two groups. (Data are shown as mean±s.e.m., P value is indicated on the figures, one sample t-test versus hypothetical mean 1, n=5).


( a ) gross anatomical views of representative 4 % paraformaldehyde - fixed livers of liver index . ( Data are shown as mean ± s . e . m . , P values are indicated on figures , unpaired Student ' s t - , n = 3 mice each group ) . Scale bar , 1 cm .

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(a) The gross anatomical views of representative 4% paraformaldehyde-fixed livers and quantification of liver index. (Data are shown as mean±s.e.m., P values are indicated on the figures, unpaired Student's t-test, n=3 mice each group). Scale bar, 1 cm.


( b ) haematoxylin eosin ( HE ) CD45 of 10 - month - old WT NRBF2 KO mice livers . NRBF2 KO liver shows mild increase of ductular reaction ( yellow arrow ) isolated necrosis ( red arrows ) . necrotic foci were further confirmed by CD45 ( black arrow ) . This is a representative image from four mice per genotype . Scale bar , 50 μm . ( c ) number of necrotic foci ductular reaction scores were recorded under bright - field microscope . At least 10 randomly selected fields ( 10 × ) from each mouse four mice per genotype were analysed . ( Data are shown as mean ± s . e . m . , P values are indicated on figures . Ductular reaction scores , unpaired Student ' s t - ; necrotic foci , Mann - Whitney U - , n = 4 mice each group ) . BD , bile ; CV , central vein ; PV , portal vein .

O O O O O O O O B-MEASURED_VAR O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O 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-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) The haematoxylin and eosin (HE) and CD45 staining of 10-month-old WT and NRBF2 KO mice livers. The NRBF2 KO liver shows mild increase of ductular reaction (yellow arrow) and isolated necrosis (red arrows). The necrotic foci were further confirmed by CD45 staining (black arrow). This is a representative image from four mice per genotype. Scale bar, 50 μm. (c) The number of necrotic foci and ductular reaction scores were recorded under bright-field microscope. At least 10 randomly selected fields (10 × ) from each mouse and four mice per genotype were analysed. (Data are shown as mean±s.e.m., P values are indicated on the figures. Ductular reaction scores, unpaired Student's t-test; necrotic foci, Mann-Whitney U-test, n=4 mice each group). BD, bile duct; CV, central vein; PV, portal vein.


( a ) WB shows accumulation of p62 in liver of NRBF2 KO mice . Liver lysates were immunoprobed with antibodies indicated . p62 are upregulated in KO mouse liver . ( b ) result shows that p62 level increased more than threefold in NRBF2 KO miceliver compared with WT miceliver . ( Data are shown as mean ± s . e . m . , P values are indicated on figures , unpaired Student ' s t - ; WT , n = 5 mice ; KO , n = 4 mice ) .

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(a) WB analysis shows accumulation of p62 in the liver of NRBF2 KO mice. Liver lysates were immunoprobed with antibodies indicated. p62 protein levels are upregulated in the KO mouse liver. (b) The quantification result shows that the p62 level increased more than threefold in the NRBF2 KO miceliver compared with WT miceliver. (Data are shown as mean±s.e.m., P values are indicated on the figures, unpaired Student's t-test; WT, n=5 mice; KO, n=4 mice).


( c ) WB shows accumulation of high - molecular ( HMW ) ubiquitin in liver of NRBF2 KO mice . Insoluble fractions of liver lysates were immunoprobed with an anti - ubiquitin antibody , n = 4 mice per genotype .

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(c) WB analysis shows accumulation of high-molecular weight (HMW) ubiquitin in the liver of NRBF2 KO mice. Insoluble fractions of liver lysates were immunoprobed with an anti-ubiquitin antibody, n=4 mice per genotype.


( d ) Atg14L - linked Vps34kinase activity is impaired in mice of NRBF2 KO mice . Atg14L - linked Vps34 is pulled down by an anti - Atg14L antibody is subjected to KA . IP products inputs were immunoblotted using antibodies indicated . ( e ) of IPed Vps34kinase activity versus IPed Atg14L shows significant difference between WT NRBF2 KO miceliver . ( Data are shown as mean ± s . e . m . , P values are indicated on figure , unpaired Student ' s t - , n = 4 mice each group ) .

O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O B-CONTROLLED_VAR O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(d) Atg14L-linked Vps34kinase activity is impaired in the mice of NRBF2 KO mice. Atg14L-linked Vps34 is pulled down by an anti-Atg14L antibody and is subjected to KA. IP products and inputs were immunoblotted using the antibodies indicated. (e) Quantification of IPed Vps34kinase activity versus IPed Atg14L shows the significant difference between WT and NRBF2 KO miceliver. (Data are shown as mean±s.e.m., P values are indicated on the figure, unpaired Student's t-test, n=4 mice each group).


( f ) Focal accumulation of p62 in liver of NRBF2 KO mice . Liver frozen sections ( 15 μm in thickness ) were stained with an anti - p62 antibody . Focal accumulation of p62 is observed in KO mice liver but not in WT mice liver . Scale bars , 100 μm or 20 μm ( amplified regions ) . This is a representative image from four mice per genotype .

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(f) Focal accumulation of p62 in the liver of NRBF2 KO mice. Liver frozen sections (15 μm in thickness) were stained with an anti-p62 antibody. Focal accumulation of p62 is observed in the KO mice liver but not in the WT mice liver. Scale bars, 100 μm or 20 μm (amplified regions). This is a representative image from four mice per genotype.


( g ) Accumulated p62 colocalizes with NQO1 . Liver frozen sections were double stained with an anti - p62 antibody an anti - NQO1 antibody . This is a representative image from four mice per genotype . Scale bars , 20 μm .

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(g) Accumulated p62 colocalizes with NQO1. Liver frozen sections were double stained with an anti-p62 antibody and an anti-NQO1 antibody. This is a representative image from four mice per genotype. Scale bars, 20 μm.


WT NRBF2 KO MEFs were treated with DMSO , 1 μM thapsigargin ( TP ) or 1 μM tunicamycin ( TN ) for 24 or 48 h . ( a ) Then were either imaged under phase - contrast microscope or ( b ) stained with propidium iodide ( PI ) subjected to rate by flow cytometry . representative of after treatments quantitative of PI - positive rate show marked increase of in NRBF2 KO MEFs compared with WT MEFs . ( Data are shown as mean ± s . e . m . , P values are indicated on figure , unpaired Student ' s t - , n = 5 ) . Scale bars , 100 μm ; Con , control .

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WT and NRBF2 KO MEFs were treated with DMSO, 1 μM thapsigargin(TP) or 1 μM tunicamycin (TN) for 24 or 48 h. (a) Then cells were either imaged under phase-contrast microscope or (b) stained with propidium iodide (PI) and subjected to cell death rate analysis by flow cytometry. The representative images of cell morphology after treatments and quantitative analysis of PI-positive cells rate show marked increase of cell death in the NRBF2 KO MEFs compared with WT MEFs. (Data are shown as mean±s.e.m., P values are indicated on the figure, unpaired Student's t-test, n=5). Scale bars, 100 μm; Con, control.


( c ) Re - introducing NRBF2 to NRBF2 KO MEFs reverses vulnerability of to ER stress . NRBF2 - CFP stably expressing were established in NRBF2 KO background . were treated with DMSO or 1 μM TP for 24 h then stained with PI subjected to rate by flow cytometry . rate in CFP - positive ( + ) population ( NRBF2 - CFP expression ) negative ( − ) population ( no NRBF2 - CFP expression ) were recorded analysed , respectively . ( Data are shown as mean ± s . e . m . , P values are indicated on figure , unpaired Student ' s t - , n = 3 ) .

O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O 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-MEASURED_VAR O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(c) Re-introducing NRBF2 to NRBF2 KO MEFs reverses the vulnerability of cells to ER stress. NRBF2-CFP stably expressing cells were established in the NRBF2 KO background. Cells were treated with DMSO or 1 μM TP for 24 h then stained with PI and subjected to cell death rate analysis by flow cytometry. The cell death rate in CFP-positive (+) population (NRBF2-CFP expression) and negative (−) population (no NRBF2-CFP expression) were recorded and analysed, respectively. (Data are shown as mean±s.e.m., P values are indicated on the figure, unpaired Student's t-test, n=3).


( d ) ER stress inducers trigger caspase - 8 caspase - 3 activation in NRBF2 KO MEFs . MEFs were treated with DMSO , 1 μM TP or 1 μM TN for 24 h , then immunoprobed with indicated antibodies . Marked accumulation of caspase - 8 ( C - Cas 8 ; p43 p18 units ) caspase - 3 ( C - Cas 3 ) are observed in NRBF2 KO MEFs but not WT MEFs treated with TP or TN . This is a representative image from three independent experiments .

O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O

(d) ER stress inducers trigger caspase-8 and caspase-3 activation in NRBF2 KO MEFs. MEFs were treated with DMSO, 1 μM TP or 1 μM TN for 24 h, and then immunoprobed with indicated antibodies. Marked accumulation of cleaved caspase-8 (C-Cas 8; p43 and p18 units) and cleaved caspase-3 (C-Cas 3) are observed in the NRBF2 KO MEFs but not WT MEFs treated with TP or TN. This is a representative image from three independent experiments.


( e ) C - Cas 8 preferentially localizes at p62 aggregation foci . MEFs were treated with DMSO or 1 μM TP for 24 h , then co - stained with an anti - p62 antibody an anti - caspase - 8 antibody . C - Cas 8 signals are triggered in NRBF2 KO MEFs treated with TP preferentially localize at p62 aggregation foci . Scale bars , 20 μm . This is a representative image from three independent experiments .

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(e) C-Cas 8 preferentially localizes at p62 aggregation foci. MEFs were treated with DMSO or 1 μM TP for 24 h, and then co-stained with an anti-p62 antibody and an anti-cleaved caspase-8 antibody. The C-Cas 8 fluorescence signals are triggered in NRBF2 KO MEFs treated with TP and preferentially localize at p62 aggregation foci. Scale bars, 20 μm. This is a representative image from three independent experiments.


( a ) NRBF2 is critical for assembly of Vps34 - Vps15 Atg14L - Beclin 1 . Brain lysate from WT NRBF2 KO mice were subjected to co - IP by Vps34 antibody Atg14L antibody . IP products were subjected to in vitro lipid KA WB using indicated antibodies . ( b , c ) results shows that Vps34 - Vps15 Atg14L - Beclin 1 interactions as well as Atg14L - linked Vps34 kinase activity are markedly impaired in NRBF2 KO mice . ( Data are shown as mean ± s . e . m . , P values are indicated on figures , unpaired Student ' s t - , n = 4 mice in each group ; NS , not significant ) .

O O O B-CONTROLLED_VAR O O O O O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR 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-MEASURED_VAR O B-MEASURED_VAR B-CONTROLLED_VAR O B-MEASURED_VAR I-MEASURED_VAR O O O O B-CONTROLLED_VAR O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(a) NRBF2 is critical for the assembly of Vps34-Vps15 and Atg14L-Beclin 1 complex. Brain lysate from WT and NRBF2 KO mice were subjected to co-IP by Vps34 antibody and Atg14L antibody. The IP products were subjected to in vitro lipid KA and WB analysis using the indicated antibodies. (b,c) Quantification results shows that Vps34-Vps15 and Atg14L-Beclin 1 complex interactions as well as Atg14L-linked Vps34 kinase activity are markedly impaired in NRBF2 KO mice. (Data are shown as mean±s.e.m., P values are indicated on the figures, unpaired Student's t-test, n=4 mice in each group; NS, not significant).


( d ) Overexpression of NRBF2 enhances overall Vps34kinase activity . HEK293T were co - transfected with Myc - Vps34 - Vps15 - His CFP , NRBF2 - CFP , dMIT - CFP or dCCD - CFP . Total Vps34 was pulled down by an anti - myc antibody subjected to KA . IP products inputs are immunoblotted using antibodies indicated . ( e ) of IPed Vps34kinase activity , Vps15 normalized with IPed Vps34 shows that NRBF2 - CFP , but not CFP , dMIT - CFP or dCCD - CFP significantly enhance Vps34kinase activity Vps34 - Vps15 interaction . ( Data are shown as mean ± s . e . m . , P values are indicated on figures . IPed Vps15 / IPed Vps34 , one sample t - versus hypothetical mean 1 , Bonferroni correction , n = 3 ; IPed Vps34kinase activity / IPed Vps34 , multiple t - followed by Bonferroni correction , n = 3 ) .

O O O O O B-CONTROLLED_VAR O O B-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O B-MEASURED_VAR O O O 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-MEASURED_VAR O O B-MEASURED_VAR O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR 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-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O

(d) Overexpression of NRBF2 enhances overall Vps34kinase activity. HEK293T cells were co-transfected with Myc-Vps34-Vps15-His and CFP, NRBF2-CFP, dMIT-CFP or dCCD-CFP. Total Vps34 was pulled down by an anti-myc antibody and subjected to KA. IP products and inputs are immunoblotted using the antibodies indicated. (e) Quantification of IPed Vps34kinase activity, Vps15 protein normalized with IPed Vps34 shows that NRBF2-CFP, but not CFP, dMIT-CFP or dCCD-CFP significantly enhance Vps34kinase activity and Vps34-Vps15 interaction. (Data are shown as mean±s.e.m., P values are indicated on the figures. IPed Vps15/IPed Vps34, one sample t-test versus hypothetical mean 1, Bonferroni correction, n=3; IPed Vps34kinase activity/IPed Vps34, multiple t-test followed by Bonferroni correction, n=3).


A NHF , Lowe 1676 Lowe 3265 were grown in complete media or 5 h serum starvation , then immunostained with LAMP1 antibody . Dashed lines in outline boundaries . Scale bar , 10 μm . C Quantitative analyses of lysosome perinuclear distribution in experiments shown in A . intracellular distribution of LAMP1 positive vesicles was quantified as described in Methods . n = 3 per group in which 30 were counted per condition per experiment . Data information : data are presented as mean ± . * ≤ 0 . 05 , * * ≤ 0 . 01 , * * * ≤ 0 . 001 , * * * * P ≤ 0 . 0001 , n . s . , not significant , calculated by two - Student ' s t - .

O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O 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-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A NHF, Lowe 1676 and Lowe 3265 cells were grown in complete media or 5 h serum starvation, then immunostained with LAMP1 antibody. Dashed lines in images outline cell boundaries. Scale bar, 10 μm. C Quantitative analyses of lysosome perinuclear distribution in the experiments shown in A. The intracellular distribution of LAMP1 positive vesicles was quantified as described in the Methods. n = 3 per group in which 30 cells were counted per condition per experiment. Data information: data are presented as mean ± SEM. * ≤ 0.05, ** ≤ 0.01, ***≤ 0.001, ****P ≤ 0.0001, n.s., not significant, calculated by two-tailed Student's t-test.


B Wildtype ( Ocrl + / y ) Ocrl - / - MEF were grown in complete media or 5 h serum starvation , then immunostained with indicated antibodies . Dashed lines in outline boundaries . Scale bar , 10 μm .

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B Wildtype (Ocrl+/y) and Ocrl-/- MEF cells were grown in complete media or 5 h serum starvation, then immunostained with indicated antibodies. Dashed lines in images outline cell boundaries. Scale bar, 10 μm.


D , E Quantitative analyses of lysosome mTOR perinuclear distribution in experiments shown in B . intracellular distribution of mTOR positive vesicles was quantified as described in Methods . n = 3 per group in which 30 were counted per condition per experiment . Data information : , data are presented as mean ± . * ≤ 0 . 05 , * * ≤ 0 . 01 , * * * ≤ 0 . 001 , * * * * P ≤ 0 . 0001 , n . s . , not significant , calculated by two - Student ' s t - .

O O O O O O O B-MEASURED_VAR O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D, E Quantitative analyses of lysosome and mTOR perinuclear distribution in the experiments shown in B. The intracellular distribution of mTOR positive vesicles was quantified as described in the Methods. n = 3 per group in which 30 cells were counted per condition per experiment. Data information: , data are presented as mean ± SEM. * ≤ 0.05, ** ≤ 0.01, ***≤ 0.001, ****P ≤ 0.0001, n.s., not significant, calculated by two-tailed Student's t-test.


I Snap of WT ( Ocrl + / y ) , OCRL knockout ( Ocrl - / - ) IOB MEFs transfected with lysosome - GFP . After transfection , WT , OCRL knockout IOB MEFs were left in serum starvation for 8 h . Scale bar , 5 μm . J Quantitative analyses of tubulated lysosomes in experiments shown in H . n = 3 per group in which 10 were counted per condition per experiment . D Data information : data are presented as mean ± . * ≤ 0 . 05 , * * ≤ 0 . 01 , * * * ≤ 0 . 001 , * * * * P ≤ 0 . 0001 , n . s . , not significant , calculated by two - Student ' s t - .

O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

I Snap images of WT (Ocrl+/y), OCRL knockout (Ocrl-/-) and IOB MEFs transfected with lysosome-GFP. After transfection, WT, OCRL knockout and IOB MEFs were left in serum starvation for 8 h. Scale bar, 5 μm. J Quantitative analyses of tubulated lysosomes in the experiments shown in H. n = 3 per group in which 10 cells were counted per condition per experiment. D Data information: data are presented as mean ± SEM. * ≤ 0.05, ** ≤ 0.01, ***≤ 0.001, ****P ≤ 0.0001, n.s., not significant, calculated by two-tailed Student's t-test.


A Immunoblot of endogenous p - mTOR ( Ser2448 ) , total mTOR , p - p70 S6K ( Thr389 ) total p70 S6K in NHF , Lowe 1676 Lowe 3265 grown in complete media or 5 h serum starvation . p - p70 S6K ( Thr389 ) over total p70 S6K ratios normalized to NHF , three independent experiments . B Immunoblot of endogenous p - mTOR ( Ser2448 ) , total mTOR , p - p70 S6K ( Thr389 ) total p70 S6K in WT ( Ocrl + / y ) IOB MEFs grown in complete medium or 5 h serum starvation . p - p70 S6K ( Thr389 ) over total p70 S6K ratios normalized to NHF , three independent experiments . C Data information : data are presented as mean ± . * ≤ 0 . 05 , * * ≤ 0 . 01 , * * * ≤ 0 . 001 , * * * * P ≤ 0 . 0001 , n . s . , not significant , calculated by two - Student ' s t - unless otherwise stated .

O O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR I-MEASURED_VAR O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR I-MEASURED_VAR O O O O B-MEASURED_VAR I-MEASURED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A Immunoblot analysis of endogenous p-mTOR (Ser2448), total mTOR, p-p70 S6K (Thr389) and total p70 S6K in NHF, Lowe 1676 and Lowe 3265 cells grown in complete media or 5 h serum starvation. p-p70 S6K (Thr389) over total p70 S6K ratios normalized to NHF cells, three independent experiments. B Immunoblot analysis of endogenous p-mTOR (Ser2448), total mTOR , p-p70 S6K (Thr389) and total p70 S6K in WT (Ocrl+/y) and IOB MEFs grown in complete medium or 5 h serum starvation. p-p70 S6K (Thr389) over total p70 S6K ratios normalized to NHF cells, three independent experiments. C Data information: data are presented as mean ± SEM. * ≤ 0.05, ** ≤ 0.01, ***≤ 0.001, ****P ≤ 0.0001, n.s., not significant, calculated by two-tailed Student's t-test unless otherwise stated.


D NHF Lowe 1676 were grown in complete media , - acid ( aa ) / FBS starved for 5 h , or starved then recovered in - acid / FBS - containing medium , then immunoblotted using antibodies against endogenous p - mTOR ( Ser2448 ) , total mTOR , p - p70 S6K ( Thr389 ) total p70 S6K . Data information : data are presented as mean ± . * ≤ 0 . 05 , * * ≤ 0 . 01 , * * * ≤ 0 . 001 , * * * * P ≤ 0 . 0001 , n . s . , not significant , calculated by two - Student ' s t - unless otherwise stated .

O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR I-MEASURED_VAR O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D NHF and Lowe 1676 cells were grown in complete media, amino-acid (aa)/FBS starved for 5 h, or starved and then recovered in amino-acid/FBS-containing medium, then immunoblotted using antibodies against endogenous p-mTOR (Ser2448), total mTOR, p-p70 S6K (Thr389) and total p70 S6K. Data information: data are presented as mean ± SEM. * ≤ 0.05, ** ≤ 0.01, ***≤ 0.001, ****P ≤ 0.0001, n.s., not significant, calculated by two-tailed Student's t-test unless otherwise stated.


A Representative of NHF , Lowe 1676 Lowe 3265 immunostained with anti - α - tubulin ( red ) anti - γ - tubulin ( green ) antibodies . Scale bar , 10 μm . B Quantitative analyses of focused microtubule arrays numbers at γ - tubulin ( microtubule organizing center MTOC ) measured in experiments shown in A . n = 3 per group , three independent experiments . C Data information : data are presented as mean ± . * ≤ 0 . 05 , * * ≤ 0 . 01 , calculated by two - way ANOVA , multiple comparisons .

O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A Representative images of NHF, Lowe 1676 and Lowe 3265 cells immunostained with anti-α-tubulin (red) and anti-γ-tubulin (green) antibodies. Scale bar, 10 μm. B Quantitative analyses of focused microtubule arrays numbers at γ-tubulin (microtubule organizing center MTOC) measured in the experiments shown in A. n = 3 cells per group, three independent experiments. C Data information: data are presented as mean ± SEM. * ≤ 0.05, ** ≤ 0.01, calculated by two-way ANOVA, multiple comparisons.


C WT ( Ocrl + / y ) , OCRL knockout ( Ocrl - / - ) IOB MEFs derived from corresponding mice were costained with anti - α - tubulin anti - γ - tubulin antibodies . Nuclei were stained with DAPI ( blue ) . Scale bar , 10 μm . D Quantitative analyses of focused microtubule arrays numbers at γ - tubulin ( microtubule organizing center MTOC ) measured in experiments shown in C . n = 3 per group in which 30 were counted per condition per experiment . E Data information : data are presented as mean ± . * ≤ 0 . 05 , * * ≤ 0 . 01 , calculated by two - way ANOVA , multiple comparisons .

O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C WT (Ocrl+/y), OCRL knockout (Ocrl-/-) and IOB MEFs derived from the corresponding mice were costained with anti-α-tubulin and anti- γ-tubulin antibodies. Nuclei were stained with DAPI (blue). Scale bar, 10 μm. D Quantitative analyses of focused microtubule arrays numbers at γ-tubulin (microtubule organizing center MTOC) measured in the experiments shown in C. n = 3 per group in which 30 cells were counted per condition per experiment. E Data information: data are presented as mean ± SEM. * ≤ 0.05, ** ≤ 0.01, calculated by two-way ANOVA, multiple comparisons.


E Microtubule regrowth was performed on NHF , Lowe 1676 Lowe 3265 with 20 μM nocodazole for 2 h on ice , followed by rinse with PBS , addition of pre - warmed medium to induce microtubule regrowth . were then immunostained with anti - α - tubulin anti - γ - tubulin antibodies at indicated time point after addition of pre - warmed medium . magnified view shows microtubules organization center . Scale bar , 10 μm . F Quantitative analyses of focused microtubule arrays numbers at γ - tubulin ( microtubule organizing center , MTOC ) measured in experiments shown in E . n = 3 per group in which 30 were counted per condition per experiment . D Data information : data are presented as mean ± . * ≤ 0 . 05 , * * ≤ 0 . 01 , calculated by two - way ANOVA , multiple comparisons .

O O O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E Microtubule regrowth assay was performed on NHF, Lowe 1676 and Lowe 3265 cells with 20 μM nocodazole for 2 h on ice, followed by rinse with PBS, and addition of pre-warmed medium to induce microtubule regrowth. Cells were then immunostained with anti-α-tubulin and anti-γ-tubulin antibodies at the indicated time point after addition of pre-warmed medium. The magnified view shows the cytoplasmic microtubules organization center. Scale bar, 10 μm. F Quantitative analyses of focused microtubule arrays numbers at γ-tubulin (microtubule organizing center, MTOC) measured in the experiments shown in E. n = 3 per group in which 30 cells were counted per condition per experiment. D Data information: data are presented as mean ± SEM. * ≤ 0.05, ** ≤ 0.01, calculated by two-way ANOVA, multiple comparisons.


E Super - resolution of centrosome stained with anti - CEP164 ( red , labeling distal appendages of mother centriole ) anti - OCRL ( blue ) were acquired using Centrin2 - GFP - RPE - 1 by DeltaVision OMX V4 BLAZE system . Scale bar , 0 . 5 μm . F Super - resolution of centrosome stained with anti - CNAP1 ( red , labeling proximal ends of centriole ) anti - OCRL ( blue ) were acquired using Centrin2 - GFP - RPE - 1 by DeltaVision OMX V4 BLAZE system . Scale bar , 0 . 5 μm . G

O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O 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-MEASURED_VAR O O O O O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O

E Super-resolution images of centrosome stained with anti-CEP164 (red, labeling distal appendages of mother centriole) and anti-OCRL (blue) were acquired using Centrin2-GFP-RPE-1 cells by DeltaVision OMX V4 BLAZE system. Scale bar, 0.5 μm. F Super-resolution images of centrosome stained with anti-CNAP1 (red, labeling proximal ends of centriole) and anti-OCRL (blue) were acquired using Centrin2-GFP-RPE-1 cells by DeltaVision OMX V4 BLAZE system. Scale bar, 0.5 μm. G


H Representative of co - localization of OCRL - WT , GFP - OCRL - △ RhoGAP GFP - OCRL - △ ASH + RhoGAP with γ - tubulin . Insets , enlargements of outlined areas . Scale bars , 10 μm . I Quantitative of with without centrosomal OCRL ( centrosomal GFP signal ) in experiments shown in A . n = 3 per group in which 10 were counted per condition per experiment . Means ± , * * * * P < 0 . 0001 , n . s . , not significant , calculated by two - way ANOVA , multiple comparisons .

O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

H Representative images of the co-localization of OCRL-WT, GFP-OCRL-△RhoGAP and GFP-OCRL-△ASH+RhoGAP with γ-tubulin. Insets, enlargements of the outlined areas. Scale bars, 10 μm. I Quantitative analysis of cells with and without centrosomal OCRL (centrosomal GFP signal) in the experiments shown in A. n = 3 per group in which 10 cells were counted per condition per experiment. Means ±SEM, ****P < 0.0001, n.s., not significant, calculated by two-way ANOVA, multiple comparisons.


A RPE - 1 transfected with nontargeting control siRNA or OCRL siRNA , were left for 48 h , then subjected to IF microscopy to visualize PI ( 4 , 5 ) P2 ( green ) , γ - tubulin ( red ) nuclei ( blue , DAPI ) . Scale bar , 10 μm . B Quantitative analyses of centrosomal PI ( 4 , 5 ) P2 in experiments shown in A . n = 3 per group in which 50 were counted per condition per experiment . C Data information : data are presented as mean ± . n . s . , not significant , calculated by calculated by two - Student ' s t - .

O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A RPE-1 cells transfected with nontargeting control siRNA or OCRL siRNA, were left for 48 h, then subjected to IF microscopy to visualize PI(4,5)P2 (green), γ-tubulin (red) and nuclei (blue, DAPI staining). Scale bar, 10 μm. B Quantitative analyses of centrosomal PI(4,5)P2 fluorescence intensity in the experiments shown in A. n = 3 per group in which 50 cells were counted per condition per experiment. C Data information: data are presented as mean ± SEM. n.s., not significant, calculated by calculated by two-tailed Student's t-test.


C NHF , Lowe 1676 Lowe 3265 were subjected to IF microscopy to visualize PI ( 4 , 5 ) P2 ( green ) , gamma - tubulin ( red ) nuclei ( blue , DAPI ) . Scale bar , 10 μm . D of PI ( 4 , 5 ) P2 on centrosomes in C was measured . n = 3 per group in which 50 were counted per condition per experiment . D Data information : data are presented as mean ± . n . s . , not significant , calculated by calculated by two - Student ' s t - .

O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C NHF, Lowe 1676 and Lowe 3265 cells were subjected to IF microscopy to visualize PI(4,5)P2 (green), gamma-tubulin (red) and nuclei (blue, DAPI staining). Scale bar, 10 μm. D The fluorescence intensity of PI (4,5)P2 on centrosomes in C was measured. n = 3 per group in which 50 cells were counted per condition per experiment. D Data information: data are presented as mean ± SEM. n.s., not significant, calculated by calculated by two-tailed Student's t-test.


A SSX2IP interacts with OCRL . Plasmids carrying GFP - OCRL - WT , GFP - OCRL - △ RhoGAP GFP - OCRL - △ ASH + RhoGAP were transfected into RPE - 1 . Immunoprecipitation was performed with GFP - Trap , followed by blotting with indicated antibodies .

O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O

A SSX2IP interacts with OCRL. Plasmids carrying GFP-OCRL-WT, GFP-OCRL-△RhoGAP and GFP-OCRL-△ASH+RhoGAP were transfected into RPE-1 cells. Immunoprecipitation was performed with GFP-Trap, followed by blotting with the indicated antibodies.


D NHF , Lowe 1676 Lowe 3265 were transfected with wildtype GFP - SSX2IP or GFP - PACT - SSX2IP for 48 h , then immunostained with anti - β - tubulin ( red ) antibody . Nuclei ( blue ) were stained with DAPI . centrosomal microtubule nucleation in GFP - PACT - SSX2IP transfected was indicated by arrowhead . Scale bar , 5 μm .

O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O

D NHF, Lowe 1676 and Lowe 3265 cells were transfected with wildtype GFP-SSX2IP or GFP-PACT-SSX2IP for 48 h, then immunostained with anti-β-tubulin (red) antibody. Nuclei (blue) were stained with DAPI. The centrosomal microtubule nucleation in GFP-PACT-SSX2IP transfected cells was indicated by white arrowhead. Scale bar, 5 μm.


F Western blot of GFP , endogenous phospho - mTOR , total mTOR , in NHF Lowe 1676 . NHF Lowe 1676 were left untransfected , transfected with GFP - SSX2IP - WT or GFP - PACT - SSX2IP , then immunoblotted with indicated antibodies .

O O O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O

F Western blot analysis of GFP, endogenous phospho-mTOR, total mTOR, in NHF and Lowe 1676 cells. NHF and Lowe 1676 cells were left untransfected, transfected with GFP-SSX2IP-WT or GFP-PACT-SSX2IP, then immunoblotted with indicated antibodies.


A Z score for AUC IC50 of 37 different drugs ( shown along horizontal axis ) identified as hits in 13 of 17 samples screened . A drug hit is defined as z score of less than - 2 for both AUC IC50 . Each dot in a column represents a sample screened for that drug . of dot corresponds to IC50 z score for that sample ( larger dot , smaller IC50 ) . Dots below black horizontal represent sample with AUC z score of less than - 2 . Dots are color coded for drug hit types . color dots below black represent a hit for corresponding drug .

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

A Z score for AUC and IC50 of 37 different drugs (shown along the horizontal axis) identified as hits in 13 of 17 samples screened. A drug hit is defined as z score of less than -2 for both AUC and IC50. Each dot in a column represents a sample screened for that drug. The size of the dot corresponds to the IC50 z score for that sample (the larger the dot, the smaller the IC50). Dots below the black horizontal line represent sample with AUC z score of less than -2. Dots are color coded for drug hit types. All color dots below the black line represent a hit for the corresponding drug.


Event - free survival ( EFS ) of human CD45 + leukocytes in peripheral blood in acute lymphoblastic leukemia ( ) orthotopic models .

O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O

Event-free survival (EFS) and percentage of human CD45+ leukocytes in peripheral blood in acute lymphoblastic leukemia (ALL) orthotopic models.


EFS volume in non - CNS subcutaneous PDX models which demonstrated objective response in 1 or more treatments .

EFS and tumor volume in non-CNS subcutaneous PDX models which demonstrated objective response in 1 or more treatments.


L EFS in a CNS orthotopic model in which drug sensitivity was observed . EFS is time of inoculation of to event ( defined by neurologic symptoms or loss ) .

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

L EFS in a CNS orthotopic model in which drug sensitivity was observed. EFS is time of inoculation of tumor cells to event (defined by neurologic symptoms or weight loss).


D Treatment response by therapy tier . 14 of 29 with molecular - therapeutic options received treatment .

D Treatment response by therapy tier. 14 of 29 patients with molecular-based therapeutic options received the treatment.


C57BL / 6J mice were fed with chow diet supplemented with 0 , 0 . 5 % , 1 % SUC for 8 . Serum SUA

O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-MEASURED_VAR

Male C57BL/6J mice were fed with chow diet supplemented with 0, 0.5%, 1% SUC for 8 weeks. Serum SUA level


C57BL / 6J mice were fed with chow diet supplemented with 0 , 0 . 5 % , 1 % SUC for 8 .

O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O

Male C57BL/6J mice were fed with chow diet supplemented with 0, 0.5%, 1% SUC for 8 weeks. body weight gain


Male C57BL/6J mice were fed with chow diet supplemented with 0, 0.5%, 1% SUC for 8 weeks. fat


C57BL / 6J mice were fed with chow diet supplemented with 0 , 0 . 5 % , 1 % SUC for 8 . lean

O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O

Male C57BL/6J mice were fed with chow diet supplemented with 0, 0.5%, 1% SUC for 8 weeks. lean mass


C57BL / 6J mice were fed with chow diet supplemented with 0 , 0 . 5 % , 1 % SUC for 8 . gastrocnemius index .

O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O

Male C57BL/6J mice were fed with chow diet supplemented with 0, 0.5%, 1% SUC for 8 weeks. gastrocnemius index.


C57BL / 6J mice were fed with chow diet supplemented with 0 , 0 . 5 % , 1 % SUC for 8 . Gastrointestinal muscle fiber immunofluorescent laminin

O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-MEASURED_VAR

Male C57BL/6J mice were fed with chow diet supplemented with 0, 0.5%, 1% SUC for 8 weeks. Gastrointestinal muscle fiber immunofluorescent laminin staining


C57BL / 6J mice were fed with chow diet supplemented with 0 , 0 . 5 % , 1 % SUC for 8 . frequency histogram of fiber cross - sectional .

O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O

Male C57BL/6J mice were fed with chow diet supplemented with 0, 0.5%, 1% SUC for 8 weeks. frequency histogram of fiber cross-sectional area.


C57BL / 6J mice fed with chow diet supplemented with 0 , 0 . 5 % , 1 % SUC for 8 . muscle grip

O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O

male C57BL/6J mice fed with chow diet supplemented with 0, 0.5%, 1% SUC for 8 weeks. The muscle grip strength


C57BL / 6J mice fed with chow diet supplemented with 0 , 0 . 5 % , 1 % SUC for 8 . running time in low

O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O

male C57BL/6J mice fed with chow diet supplemented with 0, 0.5%, 1% SUC for 8 weeks. running time in low speed


C57BL / 6J mice fed with chow diet supplemented with 0 , 0 . 5 % , 1 % SUC for 8 . four - limb handing

male C57BL/6J mice fed with chow diet supplemented with 0, 0.5%, 1% SUC for 8 weeks. four-limb handing time


C57BL / 6J mice fed with chow diet supplemented with 0 , 0 . 5 % , 1 % SUC for 8 . running time in high speed .

O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O

male C57BL/6J mice fed with chow diet supplemented with 0, 0.5%, 1% SUC for 8 weeks. running time in high speed.


C57BL / 6J mice fed with chow diet supplemented with 0 , 0 . 5 % , 1 % SUC for 8 . serum of

male C57BL/6J mice fed with chow diet supplemented with 0, 0.5%, 1% SUC for 8 weeks. serum concentration of RBC


C57BL / 6J mice fed with chow diet supplemented with 0 , 0 . 5 % , 1 % SUC for 8 . HGB in whole

O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-MEASURED_VAR O O

male C57BL/6J mice fed with chow diet supplemented with 0, 0.5%, 1% SUC for 8 weeks. HGB in whole blood


C57BL / 6J mice fed with chow diet supplemented with 0 , 0 . 5 % , 1 % SUC for 8 . ex vivo gastrocnemius muscle

male C57BL/6J mice fed with chow diet supplemented with 0, 0.5%, 1% SUC for 8 weeks. ex vivo gastrocnemius muscle force


C57BL / 6J mice fed with chow diet supplemented with 0 , 0 . 5 % , 1 % SUC for 8 .

O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O

male C57BL/6J mice fed with chow diet supplemented with 0, 0.5%, 1% SUC for 8 weeks. fatigability


C57BL / 6J mice fed with chow diet supplemented with 0 , 0 . 5 % , 1 % SUC for 8 . glucose

O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-MEASURED_VAR

male C57BL/6J mice fed with chow diet supplemented with 0, 0.5%, 1% SUC for 8 weeks. glucose consumption


C57BL / 6J mice fed with chow diet supplemented with 0 , 0 . 5 % , 1 % SUC for 8 . lactate production were tested .

O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-MEASURED_VAR O O O O

male C57BL/6J mice fed with chow diet supplemented with 0, 0.5%, 1% SUC for 8 weeks. lactate production were tested.


C57BL / 6J mice were fed with chow diet supplemented with 0 , 0 . 5 % , 1 % SUC for 8 . mRNA expression of MyHC I , MyHC IIa , PGC - 1α , myoglobin , TnnT1 MyHC IIb , MyHC IIx TnnT3 in gastrocnemius muscle ( n = 5 - 6 ) .

O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O

Male C57BL/6J mice were fed with chow diet supplemented with 0, 0.5%, 1% SUC for 8 weeks. mRNA expression of MyHC I, MyHC IIa, PGC-1α, myoglobin, TnnT1 MyHC IIb, MyHC IIx and TnnT3 in the gastrocnemius muscle (n=5-6).


C57BL / 6J mice were fed with chow diet supplemented with 0 , 0 . 5 % , 1 % SUC for 8 . Immunoblots of MyHC - I , IIa IIb expression in gastrocnemius ( n = 3 - 4 ) .

O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O

Male C57BL/6J mice were fed with chow diet supplemented with 0, 0.5%, 1% SUC for 8 weeks. Immunoblots and quantification of MyHC-I, IIa and IIb protein expression in gastrocnemius (n=3-4).


C57BL / 6J mice were fed with chow diet supplemented with 0 , 0 . 5 % , 1 % SUC for 8 . Representative of Laminin ( green ) , MyHC - I IIb immunofluorescent ( red ) in gastrocnemius ( n = 3 ) . Scale bar in ( C ) represents 100 µm .

O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O

Male C57BL/6J mice were fed with chow diet supplemented with 0, 0.5%, 1% SUC for 8 weeks. Representative images and quantification of Laminin (green), MyHC-I and IIb immunofluorescent staining (red) in gastrocnemius (n=3). Scale bar in (C) represents 100 µm.


C57BL / 6J mice were fed with chow diet supplemented with 0 and1 % SUC for 6 . O2 consumption ( VO2 ) respiratory exchange ( RER )

O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-MEASURED_VAR O O O O O O O O O

Male C57BL/6J mice were fed with chow diet supplemented with 0 and1% SUC for 6 weeks. The O2 consumption (VO2) and respiratory exchange ratio (RER)


C57BL / 6J mice were fed with chow diet supplemented with 0 and1 % SUC for 6 . Serum of ( E ) NEFA in whole blood .

O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-MEASURED_VAR O O O O

Male C57BL/6J mice were fed with chow diet supplemented with 0 and1% SUC for 6 weeks. Serum concentration of (E) NEFA in whole blood.


C57BL / 6J mice were fed with chow diet supplemented with 0 and1 % SUC for 6 . enzymes activity of ( F ) SDH , ( G ) HK , ( H ) LDH in gastrocnemius .

O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O

Male C57BL/6J mice were fed with chow diet supplemented with 0 and1% SUC for 6 weeks. The enzymes activity of (F) SDH, (G) HK, and (H) LDH in gastrocnemius.


C57BL / 6J mice were fed with chow diet supplemented with 0 and1 % SUC for 6 . Immunoblots of of p - AMPK ， PGC - 1α myoglobin in gastrocnemius .

O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O

Male C57BL/6J mice were fed with chow diet supplemented with 0 and1% SUC for 6 weeks. Immunoblots and quantification of of p-AMPK，PGC-1α and myoglobin in gastrocnemius.


C57BL / 6J mice were fed with chow diet supplemented with 0 and1 % SUC for 6 . of mitochondrial electron transport chain ( ETC ) related genes expression in gastrocnemius .

O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O

Male C57BL/6J mice were fed with chow diet supplemented with 0 and1% SUC for 6 weeks. Quantification of mitochondrial and electron transport chain (ETC) related genes expression in gastrocnemius.


C57BL / 6J mice were fed with chow diet supplemented with 0 and1 % SUC for 6 . OCRs were measured under basal condition in gastrocnemius .

O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O

Male C57BL/6J mice were fed with chow diet supplemented with 0 and1% SUC for 6 weeks. OCRs were measured under basal condition in gastrocnemius.


C2C12 were treated with 0 , 0 . 5 2 mM SUC for 48 h . Representative of MyHC - I IIb immunofluorescent ( green ) in C2C12 ( n = 16 ) .

O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O

C2C12 cells were treated with 0, 0.5 and 2 mM SUC for 48 h. Representative images of MyHC-I and IIb immunofluorescent staining (green) in C2C12 cells (n=16).


C2C12 were treated with 0 , 0 . 5 2 mM SUC for 48 h . of MyHC - I IIb immunofluorescent ( green ) in C2C12 ( n = 16 ) .

O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O

C2C12 cells were treated with 0, 0.5 and 2 mM SUC for 48 h. quantification of MyHC-I and IIb immunofluorescent staining (green) in C2C12 cells (n=16).


C2C12 were treated with 0 , 0 . 5 2 mM SUC for 48 h . enzymes activity of ( C ) SDH , ( D )

O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O

C2C12 cells were treated with 0, 0.5 and 2 mM SUC for 48 h. The enzymes activity of (C) SDH, (D) LDH


C2C12 were treated with 0 , 0 . 5 2 mM SUC for 48 h . lactate production in C2C12 .

O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-MEASURED_VAR O O O O

C2C12 cells were treated with 0, 0.5 and 2 mM SUC for 48 h. lactate production in C2C12 cells.


C2C12 were treated with 0 , 0 . 5 2 mM SUC for 48 h . of mitochondrial contents in C2C12 .

O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O

C2C12 cells were treated with 0, 0.5 and 2 mM SUC for 48 h. Quantification of mitochondrial DNA contents in C2C12 cells.


C2C12 were treated with 0 , 0 . 5 2 mM SUC for 48 h . Mitochondrial electron microscopy showed ( H ) mitochondrial density , ( I ) mitochondrial coverage , ( J ) average mitochondrial in C2C12 . Scale bar in ( A ) represents 50 μm , scale bar in ( G ) represents 0 . 5 μm .

O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C2C12 cells were treated with 0, 0.5 and 2 mM SUC for 48 h. Mitochondrial electron microscopy showed the (H) mitochondrial density, (I) mitochondrial coverage, and (J) average mitochondrial area in C2C12 cell. Scale bar in (A) represents 50 μm, scale bar in (G) represents 0.5 μm.


SUNCR1 expression in gastrocnemius from sedentary or post - running mice ( n = 4 ) .

B-MEASURED_VAR O O O O O O O O O O O O O O O O

SUNCR1 protein expression in the gastrocnemius from sedentary or post-running mice (n=4).


mRNA level of SUNCR1 in gastrocnemius soleus ( n = 7 - 8 ) .

O O O B-MEASURED_VAR O O O O O O O O O O O

The mRNA level of SUNCR1 in gastrocnemius and soleus (n=7-8).


[ Ca2 + ] i in C2C12 treated with 0 or 2 mM SUC ( n = 18 - 20 ) .

O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O

[Ca2+]i in C2C12 cells treated with 0 or 2 mM SUC (n=18-20).


NFAT expression in nucleus cytoplasm of gastrocnemius 0 . 5 h or 3 h after i . p . injection of 15 mg / kg succinate in C57BL / 6J mice ( n = 4 ) .

B-MEASURED_VAR 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-CONTROLLED_VAR O O O O O O O O O O O

NFAT protein expression in nucleus and cytoplasm of gastrocnemius 0.5 h or 3 h after i. p. injection of 15 mg/kg succinate in C57BL/6J mice (n=4).


SUNCR1 expression in C2C12 transfected with vector or siSUNCR1 ( n = 3 ) .

B-MEASURED_VAR O O O O O O O O O O O O O O

SUNCR1 protein expression in C2C12 cells transfected with vector or siSUNCR1 (n=3).


[ Ca2 + ] i , in vector or siSUNCR1 transfected C2C12 treated with 0 or 2 mM SUC ( n = 5 - 6 ) .

O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O

[Ca2+]i, in vector or siSUNCR1 transfected C2C12 cells treated with 0 or 2 mM SUC (n=5-6).


enzymes activity ( n = 9 - 10 ) of ( J ) HK , ( K ) LDH , ( L ) SDH in vector or siSUNCR1 transfected C2C12 treated with 0 or 2 mM SUC ( n = 5 - 6 ) .

O O O O O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O

enzymes activity (n=9-10)of (J) HK, (K) LDH, and (L) SDH in vector or siSUNCR1 transfected C2C12 cells treated with 0 or 2 mM SUC (n=5-6).


Representative of MyHC - I IIb immunofluorescent ( green ) in C2C12 ( n = 3 ) . Scale bar in ( M ) represents 50 μm .

O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O

Representative images and quantification of MyHC-I and IIb immunofluorescent staining (green) in C2C12 cells (n=3). Scale bar in (M) represents 50 μm.


C57BL / 6J or SUNCR1 KO mice were fed with chow diet supplemented with 0 or 1 % SUC for 6 . O2 consumption ( VO2 ) ,

O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-MEASURED_VAR O O O O O

Male C57BL/6J or SUNCR1 KO mice were fed with chow diet supplemented with 0 or 1% SUC for 6 weeks. The O2 consumption (VO2), RER


C57BL / 6J or SUNCR1 KO mice were fed with chow diet supplemented with 0 or 1 % SUC for 6 . muscle grip

O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O

Male C57BL/6J or SUNCR1 KO mice were fed with chow diet supplemented with 0 or 1% SUC for 6 weeks. muscle grip strength


C57BL / 6J or SUNCR1 KO mice were fed with chow diet supplemented with 0 or 1 % SUC for 6 . four - limb handing

O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O

Male C57BL/6J or SUNCR1 KO mice were fed with chow diet supplemented with 0 or 1% SUC for 6 weeks. four-limb handing time


C57BL / 6J or SUNCR1 KO mice were fed with chow diet supplemented with 0 or 1 % SUC for 6 . low speed running time .

O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O

Male C57BL/6J or SUNCR1 KO mice were fed with chow diet supplemented with 0 or 1% SUC for 6 weeks. low speed running time.


C57BL / 6J or SUNCR1 KO mice were fed with chow diet supplemented with 0 or 1 % SUC for 6 . enzymes activity of ( H ) HK , ( I ) LDH , ( J ) SDH in gastrocnemius .

O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O

Male C57BL/6J or SUNCR1 KO mice were fed with chow diet supplemented with 0 or 1% SUC for 6 weeks. The enzymes activity of (H) HK, (I) LDH, and (J) SDH in gastrocnemius.


C57BL / 6J or SUNCR1 KO mice were fed with chow diet supplemented with 0 or 1 % SUC for 6 . Immunoblots of MyHC - I , IIb , NFAT , PGC - 1α in gastrocnemius .

O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O

Male C57BL/6J or SUNCR1 KO mice were fed with chow diet supplemented with 0 or 1% SUC for 6 weeks. Immunoblots and quantification of MyHC-I, IIb, NFAT, and PGC-1α protein in gastrocnemius.


C57BL / 6J or SUNCR1 KO mice were fed with chow diet supplemented with 0 or 1 % SUC for 6 . Representative of Laminin ( green ) , or MyHC - I IIb ( red ) immunofluorescent in gastrocnemius muscle ( n = 3 ) . Scale bar in ( M ) represents 100 µm .

O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O

Male C57BL/6J or SUNCR1 KO mice were fed with chow diet supplemented with 0 or 1% SUC for 6 weeks. Representative images and quantification of Laminin (green), or MyHC-I and IIb (red) immunofluorescent staining in gastrocnemius muscle (n=3). Scale bar in (M) represents 100 µm.


C57BL / 6J mice were injected with LV - shScrambled or shSUNCR1 lentivirus specifically into gastrocnemius at 6 of . After two of recovery , mice were fed with chow diet supplemented with 0 or 1 % SUC for 6 . SUNCR1 expression in gastrocnemius from mice transfected with shSUNCR1 lentivirus or LV - shScrambled ( n = 3 ) .

O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O

Male C57BL/6J mice were injected with LV-shScrambled or shSUNCR1 lentivirus specifically into the gastrocnemius at 6 weeks of age. After two weeks of recovery, mice were fed with chow diet supplemented with 0 or 1% SUC for 6 weeks. SUNCR1 protein expression in gastrocnemius from mice transfected with shSUNCR1 lentivirus or LV-shScrambled (n=3).


C57BL / 6J mice were injected with LV - shScrambled or shSUNCR1 lentivirus specifically into gastrocnemius at 6 of . After two of recovery , mice were fed with chow diet supplemented with 0 or 1 % SUC for 6 . running time in low

O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O

Male C57BL/6J mice were injected with LV-shScrambled or shSUNCR1 lentivirus specifically into the gastrocnemius at 6 weeks of age. After two weeks of recovery, mice were fed with chow diet supplemented with 0 or 1% SUC for 6 weeks. The running time in low speed


C57BL / 6J mice were injected with LV - shScrambled or shSUNCR1 lentivirus specifically into gastrocnemius at 6 of . After two of recovery , mice were fed with chow diet supplemented with 0 or 1 % SUC for 6 . four - limb handing

Male C57BL/6J mice were injected with LV-shScrambled or shSUNCR1 lentivirus specifically into the gastrocnemius at 6 weeks of age. After two weeks of recovery, mice were fed with chow diet supplemented with 0 or 1% SUC for 6 weeks. four-limb handing time


C57BL / 6J mice were injected with LV - shScrambled or shSUNCR1 lentivirus specifically into gastrocnemius at 6 of . After two of recovery , mice were fed with chow diet supplemented with 0 or 1 % SUC for 6 . muscle grip strength of both control gastrocnemius - specific SUNCR1 knockdown mice .

O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O

Male C57BL/6J mice were injected with LV-shScrambled or shSUNCR1 lentivirus specifically into the gastrocnemius at 6 weeks of age. After two weeks of recovery, mice were fed with chow diet supplemented with 0 or 1% SUC for 6 weeks. muscle grip strength of both control and gastrocnemius-specific SUNCR1 knockdown mice.


C57BL / 6J mice were injected with LV - shScrambled or shSUNCR1 lentivirus specifically into gastrocnemius at 6 of . After two of recovery , mice were fed with chow diet supplemented with 0 or 1 % SUC for 6 . enzymes activity of ( F ) HK , ( G ) LDH , ( H ) SDH in gastrocnemius .

O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O

Male C57BL/6J mice were injected with LV-shScrambled or shSUNCR1 lentivirus specifically into the gastrocnemius at 6 weeks of age. After two weeks of recovery, mice were fed with chow diet supplemented with 0 or 1% SUC for 6 weeks. The enzymes activity of (F) HK, (G) LDH, (H) SDH in gastrocnemius.


C57BL / 6J mice were injected with LV - shScrambled or shSUNCR1 lentivirus specifically into gastrocnemius at 6 of . After two of recovery , mice were fed with chow diet supplemented with 0 or 1 % SUC for 6 . Immunoblots of MyHC - I , IIb , NFAT , PGC - 1α in gastrocnemius ( n = 3 ) .

O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O

Male C57BL/6J mice were injected with LV-shScrambled or shSUNCR1 lentivirus specifically into the gastrocnemius at 6 weeks of age. After two weeks of recovery, mice were fed with chow diet supplemented with 0 or 1% SUC for 6 weeks. Immunoblots and quantification of MyHC-I, IIb, NFAT, and PGC-1α protein in gastrocnemius (n=3).


C Western blots of total of CHO transfected with ApCatSper 2 or 3 , non ‐ transfected control ( ‐ ) , A . punctulata sperm ( Sp ) . Western blots were probed with anti ‐ HA , anti ‐ ApCatSper 2 , or anti ‐ ApCatSper 3 antibodies . Arrows indicate bands representing ApCatSper 2 3 .

O O O O O O O O O O B-MEASURED_VAR O O O O O O O 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-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O

C Western blots of total protein of CHO cells transfected with ApCatSper 2 or 3, non‐transfected control cells (‐), and A. punctulata sperm (Sp). The Western blots were probed with anti‐HA, anti‐ApCatSper 2, or anti‐ApCatSper 3 antibodies. Arrows indicate bands representing ApCatSper 2 and 3.


D Immunocytochemistry of sperm stained with anti ‐ ApCatSper 3 , anti ‐ GC , or anti ‐ CNGK antibodies ; superposition of obtained by microscopy bright ‐ field microscopy ; scale bar = 10 μm . was stained with DAPI ( blue ) .

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D Immunocytochemistry analysis of sperm stained with anti‐ApCatSper 3, anti‐GC, or anti‐CNGK antibodies; superposition of images obtained by fluorescence microscopy and bright‐field light microscopy; scale bar = 10 μm. The DNA was stained with DAPI (blue).


E Western blot of co ‐ immunoprecipitation ( IP ) of A . punctulata sperm . input ( I ) , flow through ( FT ) , washes ( W1 ‐ 5 ) , eluate ( E ) of IP using anti ‐ ApCatSper 2 antibody were probed with anti ‐ ApCatSper 3 antibody ( upper panel ) vice versa ( lower panel ) . ApCatSper 3 ApCatSper 2 were co ‐ immunoprecipitated with anti ‐ ApCatSper 2 ( upper panel ) anti ‐ ApCatSper 3 antibody ( lower panel ) , respectively .

O O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O

E Western blot analysis of co‐immunoprecipitation (IP) of A. punctulata sperm proteins. The input (I), flow through (FT), washes (W1‐5), and the eluate (E) of the IP using the anti‐ApCatSper 2 antibody were probed with the anti‐ApCatSper 3 antibody (upper panel) and vice versa (lower panel). ApCatSper 3 and ApCatSper 2 were co‐immunoprecipitated with the anti‐ApCatSper 2 (upper panel) and anti‐ApCatSper 3 antibody (lower panel), respectively.


F by mass spectrometry of immunoprecipitated . ApCatSper 1 ‐ 4 , β , γ , δ were identified in immunoprecipitates obtained with both anti ‐ ApCatSper 2 anti ‐ ApCatSper 3 antibodies . number of tryptic peptides identified respective coverage are given .

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F Analysis by mass spectrometry of immunoprecipitated proteins. ApCatSper 1‐4, β, γ, and δ were identified in the immunoprecipitates obtained with both the anti‐ApCatSper 2 and anti‐ApCatSper 3 antibodies. The number of tryptic peptides identified and the respective sequence coverage are given.