Figure 5 ( F ) Immunofluorescence image and quantification of the expression of SOX2 in tumor tissues subjected to in vivo limiting dilution assay . Scale bar = 50 µm ( n = 3 biological replicates ; mean ± SEM ; * * P = 0 . 0024 , * * P = 0 . 0088 , and * P = 0 . 0342 ; two - tailed unpaired Student ' s t - test ) . ( I and J ) Expression levels of LSD1 in sEVs isolated from the plasma . CD9 and CD63 were used as markers of sEVs . CD9 was used as a loading control for sEV lysis ( n = 3 biological replicates ; mean ± SEM ; * P = 0 . 0256 ( patient 1 ) , * * P = 0 . 0068 ( patient 2 ) , * * P = 0 . 0013 ( patient 3 ) , * * P = 0 . 0031 ( patient 4 ) , * * * P = 0 . 0009 ( patient 5 ) , * * P = 0 . 0012 ( patient 6 ) , * * * P = 0 . 0007 ( patient 7 ) , * * * P = 0 . 0010 ( patient 8 ) , * * * P = 0 . 0001 ( patient 9 ) , and * P = 0 . 0461 ( patient 10 ) ; two - tailed unpaired Student ' s t - test ) . ( K ) Sphere formation assay results of MGC - 803 cells treated with sEVs as indicated for 7 days . Scale bar = 100 µm ( n = 3 biological replicates ; mean ± SEM ; * * P = 0 . 0019 and * * P = 0 . 0050 ; two - tailed unpaired Student ' s t - test ) .

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

Figure 5(F) Immunofluorescence image and quantification of the expression of SOX2 in tumor tissues subjected to in vivo limiting dilution assay. Scale bar = 50 µm (n = 3 biological replicates; mean ± SEM; **P = 0.0024, **P = 0.0088, and *P = 0.0342; two-tailed unpaired Student's t-test).(I and J) Expression levels of LSD1 in sEVs isolated from the plasma. CD9 and CD63 were used as markers of sEVs. CD9 was used as a loading control for sEV lysis (n = 3 biological replicates; mean ± SEM; *P = 0.0256 (patient 1), **P = 0.0068 (patient 2), **P = 0.0013 (patient 3), **P = 0.0031 (patient 4), ***P = 0.0009 (patient 5), **P = 0.0012 (patient 6), ***P = 0.0007 (patient 7), ***P = 0.0010 (patient 8), ***P = 0.0001 (patient 9), and *P = 0.0461 (patient 10); two-tailed unpaired Student's t-test).(K) Sphere formation assay results of MGC-803 cells treated with sEVs as indicated for 7 days. Scale bar = 100 µm (n = 3 biological replicates; mean ± SEM; **P = 0.0019 and **P = 0.0050; two-tailed unpaired Student's t-test).


Figure 6Proliferation assay results of MGC - 803 ( A ) , MKN - 45 ( B ) , NCI - N87 ( C ) cells treated with oxaliplatin along with indicated treatments ( n = 3 biological replicates , mean ± standard error of mean ( SEM ) ; * P = 0 . 0204 ( MGC - 803 ) , * P = 0 . 0498 ( MKN - 45 ) , * P = 0 . 0473 ( NCI - N87 ) , and * P = 0 . 0019 ( LSD1 KO MGC - 803 ) ; two - tailed unpaired Student ' s t - test ) . Representative images of tumors ( E ) and the tumor weight ( F ) of mice treated with oxaliplatin in the presence or absence of sEVs ( n = 3 biological replicates ; mean ± SEM ; * P = 0 . 0488 ; two - tailed unpaired Student ' s t - test ) .

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

Figure 6Proliferation assay results of MGC-803 (A), MKN-45 (B), NCI-N87 (C) cells treated with oxaliplatin along with indicated treatments (n = 3 biological replicates, mean ± standard error of mean (SEM); *P = 0.0204 (MGC-803), *P = 0.0498 (MKN-45), *P = 0.0473 (NCI-N87), and *P = 0.0019 (LSD1 KO MGC-803); two-tailed unpaired Student's t-test).Representative images of tumors (E) and the tumor weight (F) of mice treated with oxaliplatin in the presence or absence of sEVs (n = 3 biological replicates; mean ± SEM; *P = 0.0488; two-tailed unpaired Student's t-test).


figf1 ( a ) Expression levels of Tcfeb mRNA in tissues isolated from 24 - h - fasted ( 24 h starved ) 6 - week - old mice . Values are expressed as fold change relative to Tcfeb expression in mice fed ad libitum ( Fed ) . Bars represent mean ± s . d . for n = 5 mice ; * P ≤ 0 . 05 ; * * P ≤ 0 . 01 ; * * * P ≤ 0 . 001 . ( b ) Representative β - gal staining of liver and kidney frozen sections isolated from fed and 24 - h - fasted heterozygous Tcfeb - β - gal mice . c ) Time - course expression analysis of TFEB in wild - type HeLa cells , MEFs and hepatocytes after the addition of starvation media ( time 0 ) . Data represent mean ± s . d . for n = 3 independent experiments . ( d ) Expression levels of transfected hTFEB - FLAG , Tcfeb - β - gal fusion transcript and endogenous Tcfeb mRNAs in MEFs isolated from control ( + / + ) and heterozygous Tcfeb - β - gal / + mice . Data represent mean ± s . d . for n = 3 mice ; * P ≤ 0 . 05 . ( e ) Time - course expression analysis of TFEB mRNA in control ( Ctrl ) or TFEB - overexpressing HeLa cells during fasting and re - feeding . Primers specific for the endogenous TFEB were used . Data represent mean ± s . d . for n = 3 . ( f ) Time - course expression analysis of Tcfeb mRNA in heterozygous Tcfeb - β - gal / + or control MEFs during fasting and re - feeding . Specific primers for the endogenous Tcfeb were used . Data represent mean ± s . d . for n = 3 mice . ( g ) Expression analysis of Tcfeb mRNA in heterozygous Tcfeb - β - gal / + or control hepatocytes in fed and after 24 h fasting . Data represent mean ± s . d . for n = 3 mice . ( h ) ChIP analysis from liver of mice fed ad libitum or 24 h fasted . The CLEAR elements in the first intron of Tcfeb genomic DNA are shown as numbered black rectangles as indicated in Supplementary Table S1 . Red rectangles represent exons , and the ATG indicates the first codon ( from the mouse Tcfeb isoform b ) . The histogram shows the amount of immunoprecipitated DNA as detected by quantitative PCR assay . Values were normalized to the input and plotted as relative enrichment over a mock control . Data represent mean ± s . d of 3 independent experiments .

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

figf1(a) Expression levels of Tcfeb mRNA in tissues isolated from 24-h-fasted (24 h starved) 6-week-old mice. Values are expressed as fold change relative to Tcfeb expression in mice fed ad libitum (Fed). Bars represent mean ± s.d. for n = 5 mice; *P≤0.05; **P≤0.01; ***P≤0.001.(b) Representative β-gal staining of liver and kidney frozen sections isolated from fed and 24-h-fasted heterozygous Tcfeb- β-gal mice.c) Time-course expression analysis of TFEB in wild-type HeLa cells, MEFs and hepatocytes after the addition of starvation media (time 0). Data represent mean ± s.d. for n = 3 independent experiments.(d) Expression levels of transfected hTFEB-FLAG, Tcfeb- β-gal fusion transcript and endogenous Tcfeb mRNAs in MEFs isolated from control (+/+) and heterozygous Tcfeb- β-gal/+ mice. Data represent mean ± s.d. for n = 3 mice; *P≤0.05.(e) Time-course expression analysis of TFEB mRNA in control (Ctrl) or TFEB-overexpressing HeLa cells during fasting and re-feeding. Primers specific for the endogenous TFEB were used. Data represent mean ± s.d. for n = 3.(f) Time-course expression analysis of Tcfeb mRNA in heterozygous Tcfeb- β-gal/+ or control MEFs during fasting and re-feeding. Specific primers for the endogenous Tcfeb were used. Data represent mean ± s.d. for n = 3 mice.(g) Expression analysis of Tcfeb mRNA in heterozygous Tcfeb- β-gal/+ or control hepatocytes in fed and after 24 h fasting. Data represent mean ± s.d. for n = 3 mice.(h) ChIP analysis from liver of mice fed ad libitum or 24 h fasted. The CLEAR elements in the first intron of Tcfeb genomic DNA are shown as numbered black rectangles as indicated in Supplementary Table S1. Red rectangles represent exons, and the ATG indicates the first codon (from the mouse Tcfeb isoform b). The histogram shows the amount of immunoprecipitated DNA as detected by quantitative PCR assay. Values were normalized to the input and plotted as relative enrichment over a mock control. Data represent mean ± s.d of 3 independent experiments.


figf3 ( a ) ChIP analysis from liver of mice fed ad libitum ( fed ) or 24 h - fasted ( starved ) . CLEAR sites in the promoter region of Pgc - 1α are indicated by squares . Numbers indicate the distance ( bp ) of the binding element from the start codon . Bar graphs show the amount of immunoprecipitated DNA as detected by quantitative PCR assay . Values were normalized to the input and plotted as relative enrichment over a mock control . Bar graphs represent mean ± s . d of 3 independent experiments , * P ≤ 0 . 05 ; * * P ≤ 0 . 01 . ( c ) Luciferase activity was measured after transfecting increasing amounts of TFEB - FLAG in combination with Pgc - 1α WT or Pgc - 1α DEL plasmids . Bar graphs represent mean ± s . d . of n = 3independent experiments , * * * * P ≤ 0 . 0001 compared with mock transfected cells . ( d ) Luciferase activity was measured in cells stably overexpressing TFEB cultured in normal and starved media . Bar graphs represent mean ± s . d . of n = 3independent experiments * * P ≤ 0 . 01 compared with mock transfected cells . ( e ) Quantification of mRNA levels of PGC1α in liver and hepatocytes from control , HDAd - TFEB and Tcfeb - LiKO ( liver KO ) mice treated as indicated . Bar graphs show mean ± s . d . for n = 4 mice . * P ≤ 0 . 05 ; * * P ≤ 0 . 01 . ( f ) Expression analysis of TFEB target genes in liver of mice with indicated genotypes . Bar graphs show mean ± s . d . for n = 3 mice . * P ≤ 0 . 05 ; * * P ≤ 0 . 01 ; * * * P ≤ 0 . 001 . ( g ) Expression analysis of PGC1α / PPARα target genes in liver from either fasted or fed mice with indicated genotypes . Bar graphs show mean ± s . d . for n = 4 . * P ≤ 0 . 05 ; * * P ≤ 0 . 01 compared with the respective controls ( fed or fasted ) .

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

figf3(a) ChIP analysis from liver of mice fed ad libitum (fed) or 24 h-fasted (starved). CLEAR sites in the promoter region of Pgc-1α are indicated by squares. Numbers indicate the distance (bp) of the binding element from the start codon. Bar graphs show the amount of immunoprecipitated DNA as detected by quantitative PCR assay. Values were normalized to the input and plotted as relative enrichment over a mock control. Bar graphs represent mean ± s.d of 3 independent experiments, *P≤0.05; **P≤0.01.(c) Luciferase activity was measured after transfecting increasing amounts of TFEB-FLAG in combination with Pgc-1α WT or Pgc-1α DEL plasmids. Bar graphs represent mean ± s.d. of n = 3independent experiments, ****P≤0.0001 compared with mock transfected cells.(d) Luciferase activity was measured in cells stably overexpressing TFEB cultured in normal and starved media. Bar graphs represent mean ± s.d. of n = 3independent experiments **P≤0.01 compared with mock transfected cells.(e) Quantification of mRNA levels of PGC1α in liver and hepatocytes from control, HDAd-TFEB and Tcfeb-LiKO (liver KO) mice treated as indicated. Bar graphs show mean ± s.d. for n = 4 mice. *P≤0.05; **P≤0.01.(f) Expression analysis of TFEB target genes in liver of mice with indicated genotypes. Bar graphs show mean ± s.d. for n = 3 mice. *P≤0.05;**P≤0.01; ***P≤0.001.(g) Expression analysis of PGC1α/PPARα target genes in liver from either fasted or fed mice with indicated genotypes. Bar graphs show mean ± s.d. for n = 4. *P≤0.05; **P≤0.01 compared with the respective controls (fed or fasted).


figf4 ( a ) Oil red O staining of liver sections isolated from mice with the indicated genotype fed ad libitum and 24 h fasted . Original magnification × 40 . ( b ) Toluidin blue staining of liver sections isolated from fed and 24 - h - fasted Tcfeb - LiKO and control mice ( Tcfeb flox / flox mice ) . Arrows indicate lipid droplets . Original magnification × 100 . ( c ) Bar graphs show the quantification of the number of lipid droplets per hepatocyte from electron microscopy analysis . A representative image is shown on the right . Values are mean ± s . d . of at least 10 cells per mouse ( n = 3 mice per group ) . * P ≤ 0 . 05 . ( d ) The oxygen consumption rate ( OCR ) in primary hepatocytes isolated from control and Tcfeb - LiKO mice was measured with an XF24 analyser ( Seahorse ) before and after the addition of palmitic acid ( 0 . 2 mM ) conjugated with BSA . The vertical red line indicates the time at which palmitate was added to cells . Values are mean ± s . d . for 3 independent experiments * P ≤ 0 . 05 . ( e , f ) Total FFA ( e ) and glycerol ( f ) in the serum isolated from 6 - h - fasted Tcfeb - LiKO and control mice . Values are mean ± s . d . ( n = 5 mice per group ) * P ≤ 0 . 05 compared with controls . ( g ) Total serum ketones in fed and fasted Tcfeb - LiKO and control mice . Bars are mean ± s . d . for n = 10 mice per group . * * P ≤ 0 . 01 compared with fed control mice . ( h ) EchoMRI measurement of fat and lean mass in fed and in 24 - and 48 - h - fasted mice expressed as relative percentage to fed ( 100 % in the graph ) . Indicated values are mean ± s . d . for n = 5 mice per group . * P ≤ 0 . 05 ; * * P ≤ 0 . 01 compared with fed control mice . ( i ) Visceral fat pad mass isolated from 2 - month - old - mice with indicated genotypes . Indicated values are mean ± s . d . for n = 5 mice per group . * P ≤ 0 . 05 ; compared with fed control mice .

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

figf4(a) Oil red O staining of liver sections isolated from mice with the indicated genotype fed ad libitum and 24 h fasted. Original magnification ×40. (b) Toluidin blue staining of liver sections isolated from fed and 24-h-fasted Tcfeb-LiKO and control mice (Tcfeb flox/flox mice). Arrows indicate lipid droplets. Original magnification ×100.(c) Bar graphs show the quantification of the number of lipid droplets per hepatocyte from electron microscopy analysis. A representative image is shown on the right. Values are mean ± s.d. of at least 10 cells per mouse (n = 3 mice per group). *P≤0.05.(d) The oxygen consumption rate (OCR) in primary hepatocytes isolated from control and Tcfeb-LiKO mice was measured with an XF24 analyser (Seahorse) before and after the addition of palmitic acid (0.2 mM) conjugated with BSA. The vertical red line indicates the time at which palmitate was added to cells. Values are mean ± s.d. for 3 independent experiments *P≤0.05.(e,f) Total FFA (e) and glycerol (f) in the serum isolated from 6-h-fasted Tcfeb-LiKO and control mice. Values are mean ± s.d. (n = 5 mice per group) *P≤0.05 compared with controls.(g) Total serum ketones in fed and fasted Tcfeb-LiKO and control mice. Bars are mean ± s.d. for n = 10 mice per group. **P≤0.01 compared with fed control mice.(h) EchoMRI measurement of fat and lean mass in fed and in 24- and 48-h-fasted mice expressed as relative percentage to fed (100% in the graph). Indicated values are mean ± s.d. for n = 5 mice per group. *P≤0.05;**P≤0.01 compared with fed control mice.(i) Visceral fat pad mass isolated from 2-month-old-mice with indicated genotypes. Indicated values are mean ± s.d. for n = 5 mice per group. *P≤0.05; compared with fed control mice.


figf5 ( a ) Mice with the indicated genotype were kept on a high - fat diet ( HFD ) for 12 weeks when indicated . Gross liver morphology ( upper panel ) , haematoxylin - eosin ( H / E ) ( middle panel ) , and oil red O staining of liver sections ( bottom panel ) . Ctrl , control . ( b , c ) Bar graph showing normalized liver weights ( mean ± s . d . for n = 10 mice per group ; b ) and total lipid content in mice with indicated genotype ( mean ± s . d . for n = 10 ; c ) . * P ≤ 0 . 05 ; * * * P ≤ 0 . 001 compared with control . ( d ) Toluidin blue staining of liver sections isolated from Atg7 - LiKO mice injected with HDAd - Ctrl or HDAd - TFEB vector . ( e , f ) Bar graph showing normalized liver weights ( mean ± s . d . for n = 10 mice per group * * * P ≤ 0 . 001 ; e ) and total lipid content in mice with the indicated genotype ( mean ± s . d . for n = 5 mice per group ; * * * P ≤ 0 . 001 ; NS , not significant ; f ) . Mice injected with an empty HDAd virus behaved as wild - type untreated mice ; therefore , data are not represented in the figure .

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

figf5(a) Mice with the indicated genotype were kept on a high-fat diet (HFD) for 12 weeks when indicated. Gross liver morphology (upper panel), haematoxylin-eosin (H/E) (middle panel), and oil red O staining of liver sections (bottom panel). Ctrl, control.(b,c) Bar graph showing normalized liver weights (mean ± s.d. for n = 10 mice per group; b) and total lipid content in mice with indicated genotype (mean ± s.d. for n = 10; c). *P≤0.05;***P≤0.001 compared with control.(d) Toluidin blue staining of liver sections isolated from Atg7-LiKO mice injected with HDAd-Ctrl or HDAd-TFEB vector.(e,f) Bar graph showing normalized liver weights (mean ± s.d. for n = 10 mice per group ***P≤0.001; e) and total lipid content in mice with the indicated genotype (mean ± s.d. for n = 5 mice per group; ***P≤0.001; NS, not significant; f). Mice injected with an empty HDAd virus behaved as wild-type untreated mice; therefore, data are not represented in the figure.


figf6 ( a , b ) Body weight ( a ) and visceral fat mass ( b ) isolated from 2 - month - old - mice with the indicated genotypes . n = 5 mice per group . ( c - i ) Serum metabolic profile in HDad - TFEB mice compared with control mice . ( j , k ) Respiratory exchange ratio ( RER ; VCO2 / VO2 ; j ) and fatty acid utilization calculated from data in j ( k ) . Values are mean ± s . d ( n = 10 mice per group ) * P ≤ 0 . 05 ; * * P ≤ 0 . 01 ; compared with controls . VLDL , very - low - density lipoprotein ; HDL , high - density lipoprotein .

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

figf6(a,b) Body weight (a) and visceral fat mass (b) isolated from 2-month-old-mice with the indicated genotypes. n = 5 mice per group.(c-i) Serum metabolic profile in HDad-TFEB mice compared with control mice.(j,k) Respiratory exchange ratio (RER; VCO2/VO2; j) and fatty acid utilization calculated from data in j (k). Values are mean ± s.d (n = 10 mice per group) *P≤0.05;**P≤0.01; compared with controls. VLDL, very-low-density lipoprotein; HDL, high-density lipoprotein.


figf7 ( a ) Body weight curves of male mice fed with the high - fat diet ( HFD ; 40 % calories from fat ) for 10 weeks starting from 5 weeks of age ( 0 on the x axis ) . Mice were injected with HDAd - TFEB either 1 week before ( early inj ) , or 4 weeks after , ( late inj ) being placed on the high - fat diet , as indicated by the arrows . Values are represented as percentages of weight increase . Ctrl , control . ( b ) Whole - body composition analysis ( EchoMRI ) of the same mice as in a after 10 weeks of the high - fat diet . In a , b n = 10 mice per group ; bars represent mean ± s . d . * P ≤ 0 . 05 ; * * P ≤ 0 . 01 ; * * * P ≤ 0 . 001 compared with control high - fat diet group . ( c - f ) Total serum insulin , leptin , triglyceride and cholesterol levels in control and HDAd - TFEB mice kept on the high - fat diet for 10 weeks . Value are mean ± s . d . n = 10 add per group . * P ≤ 0 . 05 ; * * * * P ≤ 0 . 0001 compared with control . ( g - i ) Glucose and insulin tolerance tests in control and HDAd - TFEB mice challenged with the high - fat diet for 10 weeks . ( g , h ) Glucose ( g ) and serum insulin ( h ) levels at the indicated time points after glucose challenge . ( i ) Glucose levels at the indicated time points after insulin challenge . In g - i , values are mean ± s . d . n = 7 mice per group ; * P ≤ 0 . 05 ; * * P ≤ 0 . 01 compared with control . Mice injected with an empty HDAd virus behaved as wild - type untreated mice ; therefore , data are not represented in the figure .

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

figf7(a) Body weight curves of male mice fed with the high-fat diet (HFD; 40% calories from fat) for 10 weeks starting from 5 weeks of age (0 on the x axis). Mice were injected with HDAd-TFEB either 1 week before (early inj), or 4 weeks after, (late inj) being placed on the high-fat diet, as indicated by the arrows. Values are represented as percentages of weight increase. Ctrl, control. (b) Whole-body composition analysis (EchoMRI) of the same mice as in a after 10 weeks of the high-fat diet. In a,b n = 10 mice per group; bars represent mean ± s.d. *P≤0.05;**P≤0.01; ***P≤0.001 compared with control high-fat diet group.(c-f) Total serum insulin, leptin, triglyceride and cholesterol levels in control and HDAd-TFEB mice kept on the high-fat diet for 10 weeks. Value are mean ± s.d. n = 10 add per group. *P≤0.05; ****P≤0.0001 compared with control. (g-i) Glucose and insulin tolerance tests in control and HDAd-TFEB mice challenged with the high-fat diet for 10 weeks.(g,h) Glucose (g) and serum insulin (h) levels at the indicated time points after glucose challenge. (i) Glucose levels at the indicated time points after insulin challenge. In g-i, values are mean ± s.d. n = 7 mice per group; *P≤0.05;**P≤0.01 compared with control. Mice injected with an empty HDAd virus behaved as wild-type untreated mice; therefore, data are not represented in the figure.


figf8 ( a ) hlh - 30 quantitative rtPCR showing increased expression of the C . elegans TFEB gene hlh - 30 over a time course of starvation in wild - type animals followed by a rapid decrease to the basal level following re - feeding of the animals ( mean ± s . e . m . of n = 3 ) . * P ≤ 0 . 05 ( t - test , compared with wild - type at t = 0 h ) . ( b ) hlh - 30 3 ' UTR quantitative rtPCR showing expression of hlh - 30 after 12 h starvation in wild - type and hlh - 30 ( tm1978 ) animals ( mean ± s . e . m . of n = 3 ) . * P ≤ 0 . 05 ( t - test , compared with wild - type starved ) . ( c ) Quantification of oil red O stain in starved animals relative to well - fed counterparts ( mean ± s . e . m . of n = 3 ) . * * P ≤ 0 . 01 ( t - test , compared with wild - type starved ) . ( d - g ) Representative micrographs of wild - type and hlh - 30 animals after 8 h starvation and stained with oil red O . ( h - k ) Representative TEMs of wild - type and hlh - 30 animals after 24 h starvation . IEC , intestinal epithelial cell ; EPI , epidermis ; BB , brush border ; GON , gonad ; BWM , body wall muscles , aj , apical junction ; ld , lipid droplet . Scale bar , 2 μm . ( l ) Quantitative rtPCR of starvation - induced genes in wild - type and hlh - 30 animals after 12 h starvation ( means ± s . e . m of n = 3 ) . * P ≤ 0 . 05 ; * * P ≤ 0 . 01 ; * * * P ≤ 0 . 001 ( t - test , compared with wild - type starved ) . ( m , n ) hlh - 30 is required for starvation - induced lifespan extension . One representative experiment of 2 independent trials ; error bars represent mean ± s . e . m . Median survival ( MS ) ( wild - type fed ) = 10 d ; MS ( wild - type starved ) = 17 d , P0 . 0001 versus fed using the Log - rank test ; MS ( hlh - 30 fed ) = 7 d ; MS ( hlh - 30 starved ) = 9 d , P0 . 0001 versus fed . ( o ) L1 arrest assay showing survival of wild - type and hlh - 30 starved animals relative to non - starved conditions ( mean ± s . e . m . of n = 3 ) . * * * P ≤ 0 . 001 ( t - test , compared with wild - type starved ) .

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

figf8(a) hlh-30 quantitative rtPCR showing increased expression of the C. elegans TFEB gene hlh-30 over a time course of starvation in wild-type animals followed by a rapid decrease to the basal level following re-feeding of the animals (mean±s.e.m. of n = 3). *P≤0.05 (t-test, compared with wild-type at t = 0 h).(b) hlh-30 3' UTR quantitative rtPCR showing expression of hlh-30 after 12 h starvation in wild-type and hlh-30(tm1978) animals (mean±s.e.m. of n = 3). *P≤0.05 (t-test, compared with wild-type starved).(c) Quantification of oil red O stain in starved animals relative to well-fed counterparts (mean±s.e.m. of n = 3). **P≤0.01 (t-test, compared with wild-type starved).(d-g) Representative micrographs of wild-type and hlh-30 animals after 8 h starvation and stained with oil red O.(h-k) Representative TEMs of wild-type and hlh-30 animals after 24 h starvation. IEC, intestinal epithelial cell; EPI, epidermis; BB, brush border; GON, gonad; BWM, body wall muscles, aj, apical junction; ld, lipid droplet. Scale bar, 2 μm.(l) Quantitative rtPCR of starvation-induced genes in wild-type and hlh-30 animals after 12 h starvation (means ± s.e.m of n = 3). *P≤0.05; **P≤0.01; ***P≤0.001 (t-test, compared with wild-type starved).(m,n) hlh-30 is required for starvation-induced lifespan extension. One representative experiment of 2 independent trials; error bars represent mean±s.e.m. Median survival (MS) (wild-type fed) = 10 d; MS (wild-type starved) = 17 d, P0.0001 versus fed using the Log-rank test; MS (hlh-30 fed) = 7 d; MS (hlh-30 starved) = 9 d, P0.0001 versus fed.(o) L1 arrest assay showing survival of wild-type and hlh-30 starved animals relative to non-starved conditions (mean±s.e.m. of n = 3). ***P≤0.001 (t-test, compared with wild-type starved).


Figure 1B - C . Wound healing assays were performed using IBIDI ® chambers to evaluate the role of the AhR on cell migration . Images of the wound were captured using an Axio Vert . A1 inverted microscope ( Carl Zeiss ® ) at 5x magnification . The histogram represents the mean ± s . d . Wound closure was determined by measuring the distance between the edges of the wound at time 0 and 15 h ( n = 3 independent technical experiments for each cell lines or conditions ) and compared using unpaired t - tests with the Sidak - Bonferroni method . B . Results obtained with BRAFi - sensitive or resistant SK28 cells KO for the AhR in the absence of treatment . ( n = 3 independent technical experiments for each cell lines ) . C . Results obtained for the migration assay ( 0 to 15 h ) for SK28 R cells KO or not for the AhR after treatment or not with 10 μM CH - 223191 . ( n = 3 independent technical experiments for each cell lines or conditions , mean ± s . d . ) . Statistical analysis using unpaired t - tests with the Sidak - Bonferroni method has been performed between the mean of the 3 independent experiments . D . Three - dimensional spheroid growth of BRAFi - sensitive or resistant SK28 cells KO before or after knockout of AhR by CRISPR / Cas9 in the absence of treatment . Images were captured four days after implantation of the spheroids into collagen gel . ( n = 4 independent technical experiments , mean ± s . d . E . Three - dimensional spheroid growth of BRAFi - resistant SK28 cells KO before or after knockout of AhR by CRISPR / Cas9 after daily treatment with the specific AhR inhibitor CH - 223191 ( 5 μM ) for one week or in the absence of treatment . Images were captured four days after implantation of the spheroids into collagen gel . ( n = 3 independent technical experiments , mean ± s . d . ) . Statistical analysis using unpaired t - tests method has been performed with the Sidak - Bonferroni method , ( p < 0 . 01 # # , * * ; p < 0 . 001 # # # , * * * ) .

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

Figure 1B-C. Wound healing assays were performed using IBIDI® chambers to evaluate the role of the AhR on cell migration. Images of the wound were captured using an Axio Vert.A1 inverted microscope (Carl Zeiss®) at 5x magnification. The histogram represents the mean ± s.d. Wound closure was determined by measuring the distance between the edges of the wound at time 0 and 15 h (n = 3 independent technical experiments for each cell lines or conditions) and compared using unpaired t-tests with the Sidak-Bonferroni method. B. Results obtained with BRAFi-sensitive or resistant SK28 cells KO for the AhR in the absence of treatment. (n = 3 independent technical experiments for each cell lines). C. Results obtained for the migration assay (0 to 15 h) for SK28 R cells KO or not for the AhR after treatment or not with 10 μM CH-223191. (n = 3 independent technical experiments for each cell lines or conditions, mean ± s.d.). Statistical analysis using unpaired t-tests with the Sidak-Bonferroni method has been performed between the mean of the 3 independent experiments.D. Three-dimensional spheroid growth of BRAFi-sensitive or resistant SK28 cells KO before or after knockout of AhR by CRISPR/Cas9 in the absence of treatment. Images were captured four days after implantation of the spheroids into collagen gel. (n = 4 independent technical experiments, mean ± s.d. E. Three-dimensional spheroid growth of BRAFi-resistant SK28 cells KO before or after knockout of AhR by CRISPR/Cas9 after daily treatment with the specific AhR inhibitor CH-223191 (5 μM) for one week or in the absence of treatment. Images were captured four days after implantation of the spheroids into collagen gel. (n = 3 independent technical experiments, mean ± s.d.). Statistical analysis using unpaired t-tests method has been performed with the Sidak-Bonferroni method, (p<0.01 ##, **; p<0.001 ###,***).


Figure 2A . AhR Protein levels in 501Mel CTR cells and those transduced with sgRNA targeting AhR ( # 1 , # 2 ) were analyzed by western blotting . C . Three - dimensional spheroid growth of 501Mel cells . Images were captured seven days after spheroid implantation ( n = 3 independent technical experiments , mean ± s . d . ) . Each histogram represents the mean ± s . d . Comparisons with control were performed using unpaired t - tests with the Sidak - Bonferroni method , p < 0 . 001 * * * . D . AhR Protein levels in SK28R WT , KO , and KO CA - AhR cells were analyzed by western blotting . F . Three - dimensional spheroid growth of SK28 cells KO or not for AhR by CRISPR / Cas9 and rescued by the constitutive active form of AhR ( KO CA - AhR ) . Images were captured four days after spheroid implantation ( n = 3 independent technical experiments , mean ± s . d . ) . Comparisons were performed using unpaired t - tests with the Sidak - Bonferroni method .

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

Figure 2A. AhR Protein levels in 501Mel CTR cells and those transduced with sgRNA targeting AhR (#1, #2) were analyzed by western blotting.C. Three-dimensional spheroid growth of 501Mel cells. Images were captured seven days after spheroid implantation (n = 3 independent technical experiments, mean ± s.d.). Each histogram represents the mean ± s.d. Comparisons with control were performed using unpaired t-tests with the Sidak-Bonferroni method, p<0.001 ***.D. AhR Protein levels in SK28R WT, KO, and KO CA-AhR cells were analyzed by western blotting.F. Three-dimensional spheroid growth of SK28 cells KO or not for AhR by CRISPR/Cas9 and rescued by the constitutive active form of AhR (KO CA-AhR). Images were captured four days after spheroid implantation (n = 3 independent technical experiments, mean ± s.d.). Comparisons were performed using unpaired t-tests with the Sidak-Bonferroni method.


Figure 4A . At the basal level , AhR is located at the membrane layer in a protein complex that includes HSP90 , p23 , XAP , and SRC . Immunoprecipitation of SRC was performed with specific antibodies and SRC / AhR interaction analyzed by western blotting . B . Protein levels of the AhR , p - SRC ( Y416 ) , SRC , p - FAK ( Y576 / 577 ) , and FAK in the four different melanoma cell lines ( n = 3 ) . The level of BRAFi resistance corresponds to our measure of IC50 ( Vemurafenib ) for the different cell lines 501Mel ( 0 , 23uM ) , SKMel28 ( 0 , 29uM ) , M229 ( 0 , 89uM ) and M238 ( 2 , 16uM ) . These cell lines correspond to the sensitive parental cells . C . Protein levels of the AhR , p - SRC ( Y416 ) , SRC , p - FAK ( Y576 / 577 ) , and FAK in the SK28R cell line KO or not for AhR by CRISPR / Cas9 or after rescue with the activated - form of the AhR ( CA - AhR ) . D . Protein levels of AhR , p - SRC ( Y416 ) , and SRC , in the SK28R cell line after treatment with different AhR ligands for 24h ( 5 μM BaP , 5 μM indirubin , 5 μM ITE , 10 nM TCDD , and 5 μM FICZ ) . E . Protein levels of AhR , p - SRC ( Y416 ) , and SRC , in the SK28R cell line after 24 h of treatment with increasing doses of ITE .

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

Figure 4A. At the basal level, AhR is located at the membrane layer in a protein complex that includes HSP90, p23, XAP, and SRC. Immunoprecipitation of SRC was performed with specific antibodies and SRC/AhR interaction analyzed by western blotting.B. Protein levels of the AhR, p-SRC (Y416), SRC, p-FAK (Y576/577), and FAK in the four different melanoma cell lines (n=3). The level of BRAFi resistance corresponds to our measure of IC50 (Vemurafenib) for the different cell lines 501Mel (0,23uM), SKMel28 (0,29uM), M229 (0,89uM) and M238 (2,16uM). These cell lines correspond to the sensitive parental cells.C. Protein levels of the AhR, p-SRC (Y416), SRC, p-FAK (Y576/577), and FAK in the SK28R cell line KO or not for AhR by CRISPR/Cas9 or after rescue with the activated-form of the AhR (CA-AhR).D. Protein levels of AhR, p-SRC (Y416), and SRC, in the SK28R cell line after treatment with different AhR ligands for 24h (5 μM BaP, 5 μM indirubin, 5 μM ITE, 10 nM TCDD, and 5 μM FICZ).E. Protein levels of AhR, p-SRC (Y416), and SRC, in the SK28R cell line after 24 h of treatment with increasing doses of ITE.


Figure 5B . Three - dimensional spheroid growth of a 50 % / 50 % mix of SK28R WT ( in red ) and KO cells ( in green ) over five days . Cells were treated or not with SRC inhibitors : bosutinib ( 1 µM ) or dasatinib ( 1 µM ) every two days ( n = 3 independent technical experiments , mean ± s . d . ) . Comparisons with control ( DMSO ) were performed using unpaired t - tests with the Sidak - Bonferroni method , p < 0 . 001 * * * . C . Three - dimensional spheroid growth of SK28R WT ( in red ) and KO cells ( in green ) over five days . Cells were treated or not with dasatinib ( 1 µM ) in combination with ITE ( 5 μM ) or TCDD ( 20 nM ) every two days ( n = 3 independent technical experiments , mean ± s . d . ) . Comparisons with control ( DMSO ) were performed using unpaired t - tests with the Sidak - Bonferroni method , p < 0 . 05 * , p < 0 . 01 * * . Comparisons were performed using unpaired t - tests with the Sidak - Bonferroni method . D . Protein levels of AhR , p - SRC ( Y416 ) , and SRC in the SK28R cell line after 24 h of treatment with ITE ( 5 μM ) or SRC inhibitor ( dasatinib ) ( 1 μM ) . F . Expression heatmap of the median of gene expression for the various signatures ( left ) and AhR target genes ( right ) from RNAseq datasets for SK28R cells treated or not for 24 h with SRC inhibitors : bosutinib ( Bos , 1 μM ) or dasatinib ( Das , 1 μM ) . The scale corresponds to the Z scores .

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

Figure 5B. Three-dimensional spheroid growth of a 50%/50% mix of SK28R WT (in red) and KO cells (in green) over five days. Cells were treated or not with SRC inhibitors: bosutinib (1 µM) or dasatinib (1 µM) every two days (n = 3 independent technical experiments, mean ± s.d.). Comparisons with control (DMSO) were performed using unpaired t-tests with the Sidak-Bonferroni method, p<0.001 ***.C. Three-dimensional spheroid growth of SK28R WT (in red) and KO cells (in green) over five days. Cells were treated or not with dasatinib (1 µM) in combination with ITE (5 μM) or TCDD (20 nM) every two days (n = 3 independent technical experiments, mean ± s.d.). Comparisons with control (DMSO) were performed using unpaired t-tests with the Sidak-Bonferroni method, p<0.05 *, p<0.01 **. Comparisons were performed using unpaired t-tests with the Sidak-Bonferroni method.D. Protein levels of AhR, p-SRC (Y416), and SRC in the SK28R cell line after 24 h of treatment with ITE (5 μM) or SRC inhibitor (dasatinib) (1 μM).F. Expression heatmap of the median of gene expression for the various signatures (left) and AhR target genes (right) from RNAseq datasets for SK28R cells treated or not for 24 h with SRC inhibitors: bosutinib (Bos, 1 μM) or dasatinib (Das, 1 μM). The scale corresponds to the Z scores.


Figure 6B . PDX tumor volumes were measured every 2 days until reaching 1500mm3 . Values correspond to the mean + / - sem . Statistical analysis ( 2way ANOVA ) has been performed between the different experiments ( Das or Das before combo vs Dab ) at different times . Stars represent time from when tumor size is significantly lower than group treated with BRAFi alone ( Dabrafenib ) p < 0 . 05 * , p < 0 . 01 * * . C . Kaplan - Meier survival curve for MEL006R mice treated with the different drugs . Comparison of Survival Curves have been performed using the nonparametric Log - rank ( Mantel - Cox ) test .

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

Figure 6B. PDX tumor volumes were measured every 2 days until reaching 1500mm3. Values correspond to the mean +/- sem. Statistical analysis (2way ANOVA) has been performed between the different experiments (Das or Das before combo vs Dab) at different times. Stars represent time from when tumor size is significantly lower than group treated with BRAFi alone (Dabrafenib) p<0.05 *, p<0.01 **.C. Kaplan-Meier survival curve for MEL006R mice treated with the different drugs. Comparison of Survival Curves have been performed using the nonparametric Log-rank (Mantel-Cox) test.


Figure 1Week 10 COs stained for the ventricular radial glia ( vRGs ) marker CRYAB , together with WWOX and the pan - radial glia marker SOX2 . The images on the right are enlargements of the boxed area . The dashed line defines the ventricular space . Scale = 50µm ( left ) , 25µm ( right ) ( WT : n = 8 from 3 batches , KO : n = 8 from 3 batches ) . Immunofluorescent ( IF ) staining for the glutamatergic neurons marker VGLUT1 and GABAergic neurons marker GAD67 ( GAD1 ) in week 10 COs ( WT : n = 8 from 3 batches , KO : n = 8 from 3 batches , W - AAV : n = 4 from 1 batch ) . Scale = 100µm . Quantification of the images represented in figure 1E . VGLUT1 and GAD67 were quantified as the surface area covered by the staining and were normalized to the number of nuclei in each image . The boxplot represents the 1st and 3rd quartile , with its whiskers showing the minimum and maximum points and a central band representing the median . As the samples were not normally distributed , statistical significance was determined using Kruskal - Wallis test with Dunn ' s multiple comparisons test . Outliers were removed using the ROUT test ( Q = 1 % ) . Data information : n . s ( non - significant ) , * p ≤ 0 . 05 , * * * * p ≤ 0 . 0001 .

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

Figure 1Week 10 COs stained for the ventricular radial glia (vRGs) marker CRYAB, together with WWOX and the pan-radial glia marker SOX2. The images on the right are enlargements of the boxed area. The dashed line defines the ventricular space. Scale= 50µm (left), 25µm (right) (WT: n=8 from 3 batches, KO: n=8 from 3 batches).Immunofluorescent (IF) staining for the glutamatergic neurons marker VGLUT1 and GABAergic neurons marker GAD67 (GAD1) in week 10 COs (WT: n=8 from 3 batches, KO: n=8 from 3 batches, W-AAV: n=4 from 1 batch). Scale = 100µm.   Quantification of the images represented in figure 1E. VGLUT1 and GAD67 were quantified as the surface area covered by the staining and were normalized to the number of nuclei in each image. The boxplot represents the 1st and 3rd quartile, with its whiskers showing the minimum and maximum points and a central band representing the median. As the samples were not normally distributed, statistical significance was determined using Kruskal-Wallis test with Dunn's multiple comparisons test. Outliers were removed using the ROUT test (Q=1%). Data information: n.s (non-significant), *p ≤ 0.05, ****p ≤ 0.0001.


Figure 2Sample recordings from 7 - week - old hESC - derived Cerebral Organoids ( COs ) . Sample traces show visible differences in local field potential , with WWOX - KO COs ( Red ) showing increased activity compared to WT ( Blue ) in baseline condition ( Left ) and in the presence of 100µM 4AP ( Right ) . Sample recordings from 7 - week - old hESC - derived Cerebral Organoids ( COs ) . WWOX ' s coding sequence was re - introduced into week 6 WWOX - KO COs using lentiviral transduction ( lenti - WWOX ) ( F ) Immunofluorescent staining showing WWOX expression in different populations in WWOX - KO organoids following infection with lentivirus . NT = non - treated . Scale = 50µm .

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

Figure 2Sample recordings from 7-week-old hESC-derived Cerebral Organoids (COs). Sample traces show visible differences in local field potential, with WWOX-KO COs (Red) showing increased activity compared to WT (Blue) in baseline condition (Left) and in the presence of 100µM 4AP (Right).Sample recordings from 7-week-old hESC-derived Cerebral Organoids (COs). WWOX's coding sequence was re-introduced into week 6 WWOX-KO COs using lentiviral transduction (lenti-WWOX) (F) Immunofluorescent staining showing WWOX expression in different populations in WWOX-KO organoids following infection with lentivirus. NT= non-treated. Scale = 50µm .


Figure 3Week 15 and week 24 COs stained for the astrocytic and radial glia marker GFAP , and the astrocyte - specific marker S100β ( WT W15 : n = 9 from 3 batches , KO W15 : n = 16 from 3 batches , W - AAV W15 : n = 4 organoids from 1 batch . WT W24 : n = 10 from 4 batches , KO W24 : n = 9 from 3 batches , W - AAV W24 : n = 4 from 1 batch ) . Scale = 100µmIF staining of week 6 COs for astrocytic markers in the surrounding of VZs ( WT : n = 6 from 2 batches , KO : n = 6 from 2 batches ) . Scale = 100µm ( left ) , 50µm ( right ) . Staining for the DNA damage markers γH2AX and 53BP1 in the nuclei of cells in the VZ of week 6 COs at physiological conditions , together with the pan - radial glia marker SOX2 ( WT : n = 8 from 3 individual batches , KO : n = 12 from 3 individual batches , W - AAV : n = 4 from 1 batch ) . Scale = 50µm ( left ) , 25µm ( right ) . Quantification of γH2AX ( top ) and 53BP1 foci ( bottom ) in the nuclei of cells composing the innermost layer of the VZ , normalized to the total number of nuclei in this layer . The boxplot represents the 1st and 3rd quartile , with its whiskers showing the minimum and maximum points and a central band representing the median . Statistical significance was determined using one - way ANOVA with Tukey ' s multiple comparisons test ( WT : n = 8 organoids from 3 batches , KO : n = 12 organoids from 3 batches , W - AAV : n = 4 from 1 batch ) . Data information : n . s ( non - significant ) , * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * * p ≤ 0 . 0001 .

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

Figure 3Week 15 and week 24 COs stained for the astrocytic and radial glia marker GFAP, and the astrocyte-specific marker S100β (WT W15: n=9 from 3 batches, KO W15: n=16 from 3 batches, W-AAV W15: n=4 organoids from 1 batch. WT W24: n=10 from 4 batches, KO W24: n=9 from 3 batches, W-AAV W24: n=4 from 1 batch). Scale = 100µmIF staining of week 6 COs for astrocytic markers in the surrounding of VZs (WT: n=6 from 2 batches, KO: n=6 from 2 batches). Scale = 100µm (left) , 50µm (right).   Staining for the DNA damage markers γH2AX and 53BP1 in the nuclei of cells in the VZ of week 6 COs at physiological conditions, together with the pan-radial glia marker SOX2 (WT: n=8 from 3 individual batches, KO: n=12 from 3 individual batches, W-AAV: n=4 from 1 batch). Scale = 50µm (left), 25µm (right).   Quantification of γH2AX (top) and 53BP1 foci (bottom) in the nuclei of cells composing the innermost layer of the VZ, normalized to the total number of nuclei in this layer. The boxplot represents the 1st and 3rd quartile, with its whiskers showing the minimum and maximum points and a central band representing the median. Statistical significance was determined using one-way ANOVA with Tukey's multiple comparisons test (WT: n=8 organoids from 3 batches, KO: n=12 organoids from 3 batches, W-AAV: n=4 from 1 batch).  Data information: n.s (non-significant), *p ≤ 0.05, **p ≤ 0.01, ****p ≤ 0.0001.


Figure 4RNA sequencing ( RNA - seq ) of week 15 COs and transcriptome analysis ( WT : n = 2 , KO : n = 4 ) . qPCR analysis for selected Wnt target genes validating the results of the RNA - seq . Y - axis indicates relative expression fold change . Data are represented as mean ± SEM . Statistical significance was determined using one - way ANOVA with Tukey ' s multiple comparisons test ( WT : n = 4 from 1 batch , KO : n = 4 from 1 batch , W - AAV : n = 4 from 1 batch ) . Data information : * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 . 0001 . Week 16 COs were sub - fractionated into a cytoplasmic ( C ) and nuclear ( N ) fractions . The experiment was run twice with a total of 2 WT organoids and 4 KOs organoids ( 2 for each KO line ) . KAP - 1 marks the nucleus and HSP90 marks the cytoplasm . The numbers at the bottom are a quantification of the nuclear fraction of β - catenin band intensities normalized to the cytosolic fraction [ β - C ( N / C ) ] . IF staining in week 15 COs validating the decreased levels of the deep - layer cortical markers CTIP2 ( BCL11B ) and TBR1 , and superficial - layer marker SATB2 ( WT : n = 3 , KO : n = 4 , W - AAV : n = 4 ) . Scale = 50µm . Quantification of the cortical markers seen in G , normalized to the total number of nuclei . Y - axis indicates the fold change compared to the average of the WT COs . The boxplot represents the 1st and 3rd quartile , with its whiskers showing the minimum and maximum points and a central band representing the median . Statistical significance was determined using one - way ANOVA with Tukey ' s multiple comparisons test ( WT : n = 9 from 3 batches , KO : n = 16 from 3 batches , W - AAV : n = 4 organoids from 1 batch ) . Data information : * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 . 0001 .

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

Figure 4RNA sequencing (RNA-seq) of week 15 COs and transcriptome analysis (WT: n=2, KO: n=4). qPCR analysis for selected Wnt target genes validating the results of the RNA- seq. Y-axis indicates relative expression fold change. Data are represented as mean ± SEM. Statistical significance was determined using one-way ANOVA with Tukey's multiple comparisons test (WT: n=4 from 1 batch, KO: n=4 from 1 batch, W-AAV: n=4 from 1 batch). Data information: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.Week 16 COs were sub-fractionated into a cytoplasmic (C) and nuclear (N) fractions. The experiment was run twice with a total of 2 WT organoids and 4 KOs organoids (2 for each KO line). KAP-1 marks the nucleus and HSP90 marks the cytoplasm. The numbers at the bottom are a quantification of the nuclear fraction of β-catenin band intensities normalized to the cytosolic fraction [β-C (N/C)].IF staining in week 15 COs validating the decreased levels of the deep-layer cortical markers CTIP2 (BCL11B) and TBR1, and superficial-layer marker SATB2 (WT: n=3, KO: n=4, W-AAV: n=4). Scale = 50µm.   Quantification of the cortical markers seen in G, normalized to the total number of nuclei. Y-axis indicates the fold change compared to the average of the WT COs. The boxplot represents the 1st and 3rd quartile, with its whiskers showing the minimum and maximum points and a central band representing the median. Statistical significance was determined using one-way ANOVA with Tukey's multiple comparisons test (WT: n=9 from 3 batches, KO: n=16 from 3 batches, W-AAV: n=4 organoids from 1 batch).   Data information: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.


Figure 5Peripheral blood mononuclear cells ( PBMCs ) were isolated from a WOREE patient and from his healthy parents and were reprogrammed into iPSCs , and subsequently were differentiated into COs . Week 10 WSM COs stained for the progenitor marker SOX2 , neuronal marker β3 - Tubulin and WWOX ( WSM F1 : n = 2 from 1 batch , WSM M2 : n = 2 from 1 batch , WSM S2 : n = 2 from 1 batch , WSM S5 : n = 2 from 1 batch , WSM S5 W - AAV3 : n = 2 from 1 batch , WSM S5 W - AAV6 : n = 2 from 1 batch ) . Scale = 50µm . Peripheral blood mononuclear cells ( PBMCs ) were isolated from a WOREE patient and from his healthy parents and were reprogrammed into iPSCs , and subsequently were differentiated into COs . Average firing rate over 24 neurons from WSM P COs ( 4 organoids ) , 41 neurons from WSM S COs ( 3 organoids ) and 40 neurons from WSM S W - AAV organoids ( 3 organoids ) . Statistical significance was determined using one - way ANOVA with Tukey ' s multiple comparisons test . Bars represents the mean ± SEM . Data information : ns ( non - significant ) , * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 . 0001 . Peripheral blood mononuclear cells ( PBMCs ) were isolated from a WOREE patient and from his healthy parents and were reprogrammed into iPSCs , and subsequently were differentiated into COs . GAD67 and VGLUT1 immunostaining in week 10 WSM COs ( WSM F1 : n = 2 from 1 batch , WSM M2 : n = 2 from 1 batch , WSM S2 : n = 2 from 1 batch , WSM S5 : n = 2 from 1 batch , WSM S5 W - AAV3 : n = 2 from 1 batch , WSM S5 W - AAV6 : n = 2 from 1 batch ) . Scale = 50µm . Quantification of the data shown in fig 5D . Statistical significance was determined using one - way ANOVA with Tukey ' s multiple comparisons test ( WSM F1 : n = 2 from 1 batch , WSM M2 : n = 2 from 1 batch , WSM S2 : n = 2 from 1 batch , WSM S5 : n = 2 from 1 batch , WSM S5 W - AAV3 : n = 2 from 1 batch , WSM S5 W - AAV6 : n = 2 from 1 batch ) . Data information : ns ( non - significant ) , * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 . 0001 .

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

Figure 5Peripheral blood mononuclear cells (PBMCs) were isolated from a WOREE patient and from his healthy parents and were reprogrammed into iPSCs, and subsequently were differentiated into COs.   Week 10 WSM COs stained for the progenitor marker SOX2, neuronal marker β3-Tubulin and WWOX (WSM F1: n=2 from 1 batch, WSM M2: n=2 from 1 batch, WSM S2: n=2 from 1 batch, WSM S5: n=2 from 1 batch, WSM S5 W-AAV3: n=2 from 1 batch, WSM S5 W-AAV6: n=2 from 1 batch). Scale = 50µm.   Peripheral blood mononuclear cells (PBMCs) were isolated from a WOREE patient and from his healthy parents and were reprogrammed into iPSCs, and subsequently were differentiated into COs. Average firing rate over 24 neurons from WSM P COs (4 organoids), 41 neurons from WSM S COs (3 organoids) and 40 neurons from WSM S W-AAV organoids (3 organoids). Statistical significance was determined using one-way ANOVA with Tukey's multiple comparisons test. Bars represents the mean ± SEM. Data information: ns (non-significant), *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.Peripheral blood mononuclear cells (PBMCs) were isolated from a WOREE patient and from his healthy parents and were reprogrammed into iPSCs, and subsequently were differentiated into COs. GAD67 and VGLUT1 immunostaining in week 10 WSM COs (WSM F1: n=2 from 1 batch, WSM M2: n=2 from 1 batch, WSM S2: n=2 from 1 batch, WSM S5: n=2 from 1 batch, WSM S5 W-AAV3: n=2 from 1 batch, WSM S5 W-AAV6: n=2 from 1 batch). Scale = 50µm.   Quantification of the data shown in fig 5D. Statistical significance was determined using one-way ANOVA with Tukey's multiple comparisons test (WSM F1: n=2 from 1 batch, WSM M2: n=2 from 1 batch, WSM S2: n=2 from 1 batch, WSM S5: n=2 from 1 batch, WSM S5 W-AAV3: n=2 from 1 batch, WSM S5 W-AAV6: n=2 from 1 batch).  Data information: ns (non-significant), *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.


Figure 6Week 15 WSM COs stained for astrocytic markers GFAP and S100β . ( WSM F1 : n = 2 from 1 batch , WSM M2 : n = 3 from 1 batch , WSM S2 : n = 2 from 1 batch , WSM S5 : n = 2 from 1 batch , WSM S5 W - AAV3 : n = 2 from 1 batch , WSM S5 W - AAV6 : n = 2 from 1 batch ) . Scale = 50µm . Quantification of DNA damage foci , marked by γH2AX ( left ) and 53BP1 ( right ) , in the nuclei of the cells in the VZs of week 6 WSM COs . The boxplot represents the 1st and 3rd quartile , with its whiskers showing the minimum and maximum points and a central band representing the median . Statistical significance was determined using one - way ANOVA with Tukey ' s multiple comparisons test ( WSM F1 : n = 4 from 1 batch , WSM M2 : n = 4 from 1 batch , WSM S2 : n = 4 from 1 batch , WSM S5 : n = 4 from 1 batch , WSM S5 W - AAV3 : n = 4 from 1 batch , WSM S5 W - AAV6 : n = 4 from 1 batch ) . Data information : * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 . 0001 . qPCR analysis for selected Wnt target genes in week 10 WSM COs . Y - axis indicates relative expression fold change . Data are represented as mean ± SEM . Statistical significance was determined using one - way ANOVA with Tukey ' s multiple comparisons test ( WSM P : n = 6 from 1 batch , WSM S : n = 4 from 1 batch , WSM S W - AAV : n = 4 from 1 batch ) . Data information : * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 . 0001 . Week 15 WSM COs stained for the cortical layers ' markers CTIP2 and SATB2 . ( WSM F1 : n = 2 from 1 batch , WSM M2 : n = 3 from 1 batch , WSM S2 : n = 2 from 1 batch , WSM S5 : n = 2 from 1 batch , WSM S5 W - AAV3 : n = 2 from 1 batch , WSM S5 W - AAV6 : n = 2 from 1 batch ) . Scale = 50µm . Quantification of the staining presented in E . The boxplot represents the 1st and 3rd quartile , with its whiskers showing the minimum and maximum points and a central band representing the median . Statistical significance was determined using one - way ANOVA with Tukey ' s multiple comparisons test ( WSM F1 : n = 2 from 1 batch , WSM M2 : n = 3 from 1 batch , WSM S2 : n = 2 from 1 batch , WSM S5 : n = 2 from 1 batch , WSM S5 W - AAV3 : n = 2 from 1 batch , WSM S5 W - AAV6 : n = 2 from 1 batch ) . Data information : * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 . 0001 . E

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

Figure 6Week 15 WSM COs stained for astrocytic markers GFAP and S100β. (WSM F1: n=2 from 1 batch, WSM M2: n=3 from 1 batch, WSM S2: n=2 from 1 batch, WSM S5: n=2 from 1 batch, WSM S5 W-AAV3: n=2 from 1 batch, WSM S5 W-AAV6: n=2 from 1 batch). Scale = 50µm.   Quantification of DNA damage foci, marked by γH2AX (left) and 53BP1 (right), in the nuclei of the cells in the VZs of week 6 WSM COs. The boxplot represents the 1st and 3rd quartile, with its whiskers showing the minimum and maximum points and a central band representing the median. Statistical significance was determined using one-way ANOVA with Tukey's multiple comparisons test (WSM F1: n=4 from 1 batch, WSM M2: n=4 from 1 batch, WSM S2: n=4 from 1 batch, WSM S5: n=4 from 1 batch, WSM S5 W-AAV3: n=4 from 1 batch, WSM S5 W-AAV6: n=4 from 1 batch).   Data information: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. qPCR analysis for selected Wnt target genes in week 10 WSM COs. Y-axis indicates relative expression fold change. Data are represented as mean ± SEM. Statistical significance was determined using one-way ANOVA with Tukey's multiple comparisons test (WSM P: n=6 from 1 batch, WSM S: n=4 from 1 batch, WSM S W-AAV: n=4 from 1 batch). Data information: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. Week 15 WSM COs stained for the cortical layers' markers CTIP2 and SATB2. (WSM F1: n=2 from 1 batch, WSM M2: n=3 from 1 batch, WSM S2: n=2 from 1 batch, WSM S5: n=2 from 1 batch, WSM S5 W-AAV3: n=2 from 1 batch, WSM S5 W-AAV6: n=2 from 1 batch). Scale = 50µm.   Quantification of the staining presented in E. The boxplot represents the 1st and 3rd quartile, with its whiskers showing the minimum and maximum points and a central band representing the median. Statistical significance was determined using one-way ANOVA with Tukey's multiple comparisons test (WSM F1: n=2 from 1 batch, WSM M2: n=3 from 1 batch, WSM S2: n=2 from 1 batch, WSM S5: n=2 from 1 batch, WSM S5 W-AAV3: n=2 from 1 batch, WSM S5 W-AAV6: n=2 from 1 batch).  Data information: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. E


Figure 1A Normal human fibroblasts and HGPS fibroblasts were serum - starved for 48 h and stained for progerin ( red ) , acetylated tubulin ( green ) and DNA ( blue ) . The representative images show that the cell with high progerin ( inset 2 ) , but not low progerin ( inset 1 ) , displays defective cilia formation . Scale bars , 20 μm or 2 μm ( in the magnified images ) . B The progerin fluorescence intensity of the cells was measured by the Zeiss ZEN2 software ( n ≥ 148 ) . The dashed line indicates the background level of progerin . A . U . , arbitrary units . The cells were grown in the growth medium and stained for progerin ( red ) and F - actin with phalloidin ( green ) . The cell with high or low progerin expression was defined by the progerin fluorescence intensity above or below 3 . 5 x 106 ( A . U . ) , respectively . The cell spreading area was measured according to the phalloidin staining ( n ≥ 30 ) . Scale bars , 50 m . D The cells were serum - starved for 32 h to allow cilia formation and then treated with ( + ) or without ( - ) 250 nM cytochalasin D ( Cyto D ) for another 16 h before fixation . The cells were stained for progerin ( red ) , acetylated tubulin ( green ) , F - actin ( white ) , and DNA ( blue ) . The numbers of actin filaments within 60 μm2 around the cilia ( as illustrated by the circle ) from the cells under serum - starvation without Cyto D treatment were measured ( n ≥ 55 ) . Scale bars , 20 μm or 2 μm ( in the magnified insets ) . E , F The cells were subjected to immunofluorescence stain as in ( D ) . The percentage of the cells with cilia in the total counted cells ( E , n ≥ 50 ) and the cilia length ( F , n ≥ 50 ) were measured .

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

Figure 1A Normal human fibroblasts and HGPS fibroblasts were serum-starved for 48 h and stained for progerin (red), acetylated tubulin (green) and DNA (blue). The representative images show that the cell with high progerin (inset 2), but not low progerin (inset 1), displays defective cilia formation. Scale bars, 20 μm or 2 μm (in the magnified images).B The progerin fluorescence intensity of the cells was measured by the Zeiss ZEN2 software (n ≥ 148). The dashed line indicates the background level of progerin. A.U., arbitrary units.The cells were grown in the growth medium and stained for progerin (red) and F-actin with phalloidin (green). The cell with high or low progerin expression was defined by the progerin fluorescence intensity above or below 3.5 x 106 (A.U.), respectively. The cell spreading area was measured according to the phalloidin staining (n ≥ 30). Scale bars, 50 m.D The cells were serum-starved for 32 h to allow cilia formation and then treated with (+) or without (-) 250 nM cytochalasin D (Cyto D) for another 16 h before fixation. The cells were stained for progerin (red), acetylated tubulin (green), F-actin (white), and DNA (blue). The numbers of actin filaments within 60 μm2 around the cilia (as illustrated by the circle) from the cells under serum-starvation without Cyto D treatment were measured (n ≥ 55). Scale bars, 20 μm or 2 μm (in the magnified insets).E,F The cells were subjected to immunofluorescence stain as in (D). The percentage of the cells with cilia in the total counted cells (E, n ≥ 50) and the cilia length (F, n ≥ 50) were measured.


Figure 2A The skeletal ( tabialis anterior ) muscles from control ( Lmna + / + and Lmna + / - ) and Lmna - / - mice were stained for acetylated tubulin ( green ) and nucleus ( blue ) . The representative images with low and high magnification are shown . The insets are enlarged images for cilia . B The kidneys from control and Lmna - / - mice were stained for acetylated tubulin ( green ) and AQ1 ( a marker for the proximal tubules , red ) . The insets show the cilia of proximal renal tubule cells . C The uterus from control and Lmna - / - mice was stained for acetylated tubulin ( green ) and keratin 8 ( red ) . The insets show the cilia at the stroma of the uterus . D The ovary from control and Lmna - / - mice were stained for acetylated tubulin ( green ) and keratin 8 ( red ) . The insets show the cilia of the granulosa cells in the follicles of the ovary . E , F The cilia length ( E , n = 150 - 180 ) and cilia positive cells ( F , n ≥ 1197 ) in each organ from 4 - week - old Lmna - / - mice ( n = 3 ) and their littermate controls ( n = 3 ) were measured .

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

Figure 2A The skeletal (tabialis anterior) muscles from control (Lmna+/+ and Lmna+/-) and Lmna-/- mice were stained for acetylated tubulin (green) and nucleus (blue). The representative images with low and high magnification are shown. The insets are enlarged images for cilia.B The kidneys from control and Lmna-/- mice were stained for acetylated tubulin (green) and AQ1 (a marker for the proximal tubules, red). The insets show the cilia of proximal renal tubule cells.C The uterus from control and Lmna-/- mice was stained for acetylated tubulin (green) and keratin 8 (red). The insets show the cilia at the stroma of the uterus.D The ovary from control and Lmna-/- mice were stained for acetylated tubulin (green) and keratin 8 (red). The insets show the cilia of the granulosa cells in the follicles of the ovary.E, F The cilia length (E, n=150-180) and cilia positive cells (F, n ≥ 1197) in each organ from 4-week-old Lmna-/- mice (n=3) and their littermate controls (n=3) were measured.


Figure 3A RPE cells were infected with lentiviruses encoding shRNAs specific to lamin A / C ( shLamin A / C ) or luciferase ( shLuc ) . Exogenous lamin A was re - expressed in the lamin A / C - depleted cells ( shLamin A / C - Lamin A ) . An equal amount of whole cell lysates was analyzed by immunoblotting with antibodies as indicated . B The cells were serum - starved for 48 h and stained for acetylated tubulin ( green ) , γ - tubulin ( red ) and DNA ( blue ) . The depletion of lamin A / C ( shLamin A / C ) led to impaired ciliogenesis , as manifested by no cilia ( inset 1 ) or short cilia ( inset 2 ) . Scale bars , 20 μm or 2 μm ( insets ) . C The percentage of the cells with cilia in the total counted cells ( n ≥ 626 ) was measured . D The length of cilia was measured ( n ≥ 87 in each group ) . E Cells were stained for acetylated tubulin ( green ) and γ - tubulin ( red ) after various intervals of serum starvation . The percentage of cilia - positive cells at each time point ( n ≥ 372 ) was measured .

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

Figure 3A RPE cells were infected with lentiviruses encoding shRNAs specific to lamin A/C (shLamin A/C) or luciferase (shLuc). Exogenous lamin A was re-expressed in the lamin A/C-depleted cells (shLamin A/C-Lamin A). An equal amount of whole cell lysates was analyzed by immunoblotting with antibodies as indicated.B The cells were serum-starved for 48 h and stained for acetylated tubulin (green), γ-tubulin (red) and DNA (blue). The depletion of lamin A/C (shLamin A/C) led to impaired ciliogenesis, as manifested by no cilia (inset 1) or short cilia (inset 2). Scale bars, 20 μm or 2 μm (insets).C The percentage of the cells with cilia in the total counted cells (n ≥ 626) was measured.D The length of cilia was measured (n ≥ 87 in each group).E Cells were stained for acetylated tubulin (green) and γ-tubulin (red) after various intervals of serum starvation. The percentage of cilia-positive cells at each time point (n ≥ 372) was measured.


Figure 4A - C The RPE cells as described in Fig 3 were stained for paxillin ( as a marker for focal adhesion ; green ) , F - actin ( red ) and DNA ( blue ) . The representative images are shown ( A ) . Scale bars , 20 μm or 5 μm ( insets ) . The cell spreading area of the cells ( B , n ≥ 108 ) and the number of focal adhesions per cell ( C , n ≥ 30 ) were measured . D - G The cells were serum - starved for 32 h and then treated with ( + ) or without ( - ) Cyto D ( 250 nM ) for another 16 h . The cells were stained for acetylated tubulin ( white ) , pericentrin ( green ) , F - actin ( red ) and DNA ( blue ) . The representative images are shown ( D ) . The left insets show the merged signals of acetylated tubulin and pericentrin . Scale bars , 20 μm or 2 μm ( magnified images ) . The numbers of actin filaments within 60 μm2 around the basal body ( as illustrated by the circle ) from the cells under serum - starvation without Cyto D treatment were measured ( E , n ≥ 132 ) . The percentage of the cells with cilia in the total counted cells ( F , n ≥ 357 ) and the length of cilia ( G , n ≥ 126 ) were measured .

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

Figure 4A-C The RPE cells as described in Fig 3 were stained for paxillin (as a marker for focal adhesion; green), F-actin (red) and DNA (blue). The representative images are shown (A). Scale bars, 20 μm or 5 μm (insets). The cell spreading area of the cells (B, n ≥ 108) and the number of focal adhesions per cell (C, n ≥ 30) were measured.D-G The cells were serum-starved for 32 h and then treated with (+) or without (-) Cyto D (250 nM) for another 16 h. The cells were stained for acetylated tubulin (white), pericentrin (green), F-actin (red) and DNA (blue). The representative images are shown (D). The left insets show the merged signals of acetylated tubulin and pericentrin. Scale bars, 20 μm or 2 μm (magnified images). The numbers of actin filaments within 60 μm2 around the basal body (as illustrated by the circle) from the cells under serum-starvation without Cyto D treatment were measured (E, n ≥ 132). The percentage of the cells with cilia in the total counted cells (F, n ≥ 357) and the length of cilia (G, n ≥ 126) were measured.


Figure 5A An equal amount of whole cell lysates from RPE cells or those expressing shLamin A / C or luciferase ( shLuc ) was analyzed by the immunoblotting with antibodies as indicated . RPE cells were infected with lentiviruses expressing shRNAs to nesprin 2 ( shNesprine 2 , clone # 1 and # 2 ) or luciferase ( shLuc ) as a control . An equal amount of whole cell lysates was analyzed by immunoblotting with antibodies as indicated ( B ) . The cells were serum - starved for 48 h and then stained for acetylated tubulin ( white ) , pericentrin ( green ) , F - actin ( red ) and DNA ( blue ) . The representative images are shown ( C ) The left insets show the merged signals of acetylated tubulin and pericentrin . Scale bars , 20 μm or 2 μm ( magnified images ) . The numbers of F - actin stress fibers within 60 μm2 around the basal bodies ( as illustrated by the circle ) were measured ( D , n ≥ 53 ) . E , F RPE cells expressing shNesprine 2 or shLuc were serum - starved for 32 h and then treated with ( + ) or without ( - ) Cyto D for another 16 h . The percentage of the cells with cilia in the total counted cells ( E , n ≥ 197 ) and the length of cilia ( F , n ≥ 52 ) were measured .

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

Figure 5A An equal amount of whole cell lysates from RPE cells or those expressing shLamin A/C or luciferase (shLuc) was analyzed by the immunoblotting with antibodies as indicated.RPE cells were infected with lentiviruses expressing shRNAs to nesprin 2 (shNesprine 2, clone #1 and #2) or luciferase (shLuc) as a control. An equal amount of whole cell lysates was analyzed by immunoblotting with antibodies as indicated (B).The cells were serum-starved for 48 h and then stained for acetylated tubulin (white), pericentrin (green), F-actin (red) and DNA (blue). The representative images are shown (C) The left insets show the merged signals of acetylated tubulin and pericentrin. Scale bars, 20 μm or 2 μm (magnified images). The numbers of F-actin stress fibers within 60 μm2 around the basal bodies (as illustrated by the circle) were measured (D, n ≥ 53).E,F RPE cells expressing shNesprine 2 or shLuc were serum-starved for 32 h and then treated with (+) or without (-) Cyto D for another 16 h. The percentage of the cells with cilia in the total counted cells (E, n ≥ 197) and the length of cilia (F, n ≥ 52) were measured.


Figure 6A , B RPE cells expressing shRNAs specific to lamin A / C or nesprin 2 ( shNesprin 2 , clones # 1 and # 2 ) were stained for G - actin with DNase I ( green ) . Representative images are shown ( A ) . Scale bars , 20 μm . The proportion of nuclear G - actin fluorescence intensity to the whole cell was measured ( B , n ≥ 67 ) . C An equal amount of whole cell lysates from RPE cells ( control ) and those expressing shRNAs to importin 9 ( shImportin 9 , clones # 1 and # 2 ) was analyzed by immunoblotting with antibodies as indicated . D - F The cells expressing shRNAs to importin 9 were stained for G - actin ( green ) and F - actin ( red ) . The representative images are shown ( D ) . Scale bars , 20 μm or 10 μm ( insets ) . The proportion of nuclear G - actin intensity to the whole cell ( E , n > 152 ) and the cell spreading area ( F , n ≥ 139 ) were measured . G - I RPE cells and those expressing shRNAs to importin 9 were serum - starved for 32 h and then treated with ( + ) or without ( - ) Cyto D for another 16 h and then stained for acetylated tubulin ( white ) , pericentrin ( green ) and F - actin ( red ) . Representative images are shown ( G ) . The left insets show the merged signals of acetylated tubulin and pericentrin . Scale bars , 20 μm or 2 μm ( magnified images ) . The percentage of the cells with cilia ( H , n ≥ 385 ) and the length of cilia ( I , n ≥ 111 ) were measured .

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

Figure 6A,B RPE cells expressing shRNAs specific to lamin A/C or nesprin 2 (shNesprin 2, clones #1 and #2) were stained for G-actin with DNase I (green). Representative images are shown (A). Scale bars, 20 μm. The proportion of nuclear G-actin fluorescence intensity to the whole cell was measured (B, n ≥ 67).C An equal amount of whole cell lysates from RPE cells (control) and those expressing shRNAs to importin 9 (shImportin 9, clones #1 and #2) was analyzed by immunoblotting with antibodies as indicated.D-F The cells expressing shRNAs to importin 9 were stained for G-actin (green) and F-actin (red). The representative images are shown (D). Scale bars, 20 μm or 10 μm (insets). The proportion of nuclear G-actin intensity to the whole cell (E, n > 152) and the cell spreading area (F, n ≥ 139) were measured.G-I RPE cells and those expressing shRNAs to importin 9 were serum-starved for 32 h and then treated with (+) or without (-) Cyto D for another 16 h and then stained for acetylated tubulin (white), pericentrin (green) and F-actin (red). Representative images are shown (G). The left insets show the merged signals of acetylated tubulin and pericentrin. Scale bars, 20 μm or 2 μm (magnified images). The percentage of the cells with cilia (H, n ≥ 385) and the length of cilia (I, n ≥ 111) were measured.


Figure 7A RPE cells expressing shRNAs specific to luciferase ( shLuc . ) , lamin A / C , or nesprin 2 were grown in the growth medium ( + serum ) or serum - starved for 1 h ( - serum ) and then stained for Arp2 ( red ) , CEP164 ( a marker for the mother centriole , green ) and acetylated tubulin ( white ) . The representative images are shown . Note that Arp2 was found to localize exclusively at the mother centriole ( upper panels ) or at the both mother and daughter centrioles ( lower panels ) . Scale bars , 0 . 5 μm . The percentage of cells with Arp2 localized exclusively at the mother centriole in the total counted cells was measured ( n = 131 - 240 cells in each group ) . B The cells were serum - starved for 48 h and then stained for acetylated tubulin ( green ) , Arp2 ( red ) , and γ - tubulin ( white ) . The representative images are shown . Scale bars , 1 μm ( upper panels ) and 0 . 5 μm ( lower panels ) . Note that the cell with Arp2 at the both mother and daughter centrioles fails to form cilium . C The cells were serum - starved for 48 h and then stained for myosin Va ( red ) , acetylated tubulin ( green ) and γ - tubulin ( white ) . Scale bars , 0 . 5 μm . The cartoon illustrates that myosin Va - associated vesicles ( CV + PCV ) specifically accumulate at the mother centriole in most control cells ( as in the " one centriole " image ) . CV , ciliary vesicle . PCV , periciliary vesicle . The percentage of cells with myosin Va exclusively at the mother centriole in the total counted cells was measured ( n = 160 - 200 cells in each group ) .

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

Figure 7A RPE cells expressing shRNAs specific to luciferase (shLuc.), lamin A/C, or nesprin 2 were grown in the growth medium (+ serum) or serum-starved for 1 h (- serum) and then stained for Arp2 (red), CEP164 (a marker for the mother centriole, green) and acetylated tubulin (white). The representative images are shown. Note that Arp2 was found to localize exclusively at the mother centriole (upper panels) or at the both mother and daughter centrioles (lower panels). Scale bars, 0.5 μm. The percentage of cells with Arp2 localized exclusively at the mother centriole in the total counted cells was measured (n=131-240 cells in each group).B The cells were serum-starved for 48 h and then stained for acetylated tubulin (green), Arp2 (red), and γ-tubulin (white). The representative images are shown. Scale bars, 1 μm (upper panels) and 0.5 μm (lower panels). Note that the cell with Arp2 at the both mother and daughter centrioles fails to form cilium.C The cells were serum-starved for 48 h and then stained for myosin Va (red), acetylated tubulin (green) and γ-tubulin (white). Scale bars, 0.5 μm. The cartoon illustrates that myosin Va-associated vesicles (CV+PCV) specifically accumulate at the mother centriole in most control cells (as in the "one centriole" image). CV, ciliary vesicle. PCV, periciliary vesicle. The percentage of cells with myosin Va exclusively at the mother centriole in the total counted cells was measured (n=160-200 cells in each group).


Figure 8A RPE cells expressing shRNAs specific to luciferase ( shLuc ) or lamin A / C ( shLamin A / C ) were serum - starved for 48 h and then stained for acetylated tubulin ( Ac - tubulin , green ) and CEP164 ( red ) . The representative images show that the fluorescence intensity of CEP164 at the mother centriole of cilia - negative ( left column ) and cilia - positive ( right column ) basal bodies are similar . The graphs shown are the quantification of CEP164 at cilia - negative basal bodies and cilia - positive basal bodies . B The cells were serum - starved for 32 h and then treated with ( + ) or without ( - ) Cyto D for another 16 h . The cells were stained for acetylated tubulin ( Ac - tubulin , green ) and TTBK2 ( red ) . The graphs shown are the quantifications of TTBK2 intensity at cilia - negative basal bodies and cilia - positive basal bodies . C The cells as in ( B ) were stained for acetylated tubulin ( Ac - tubulin , green ) and CP110 ( red ) . The representative images show that CP110 is not removed from the mother centriole at some of cilia - negative basal bodies ( left column ) , whereas it is removed from the mother centriole at cilia - positive basal bodies ( right column ) . The graphs shown are the quantifications of CP110 - removal at cilia - negative basal bodies and cilia - positive basal bodies .

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Figure 8A RPE cells expressing shRNAs specific to luciferase (shLuc) or lamin A/C (shLamin A/C) were serum-starved for 48 h and then stained for acetylated tubulin (Ac-tubulin, green) and CEP164 (red). The representative images show that the fluorescence intensity of CEP164 at the mother centriole of cilia-negative (left column) and cilia-positive (right column) basal bodies are similar. The graphs shown are the quantification of CEP164 at cilia-negative basal bodies and cilia-positive basal bodies.B The cells were serum-starved for 32 h and then treated with (+) or without (-) Cyto D for another 16 h. The cells were stained for acetylated tubulin (Ac-tubulin, green) and TTBK2 (red). The graphs shown are the quantifications of TTBK2 intensity at cilia-negative basal bodies and cilia-positive basal bodies.C The cells as in (B) were stained for acetylated tubulin (Ac-tubulin, green) and CP110 (red). The representative images show that CP110 is not removed from the mother centriole at some of cilia-negative basal bodies (left column), whereas it is removed from the mother centriole at cilia-positive basal bodies (right column). The graphs shown are the quantifications of CP110-removal at cilia-negative basal bodies and cilia-positive basal bodies.


Figure 1 ( A ) Heatmap of tRNA profiling of MCF10A cells at 8 and 24 hours post exposure to oxidative stress ( 200μM H2O2 ) . Biological triplicate data is depicted at each time point relative to control cells . ( B ) MCF10A cells were exposed to oxidative stress ( 200μM H2O2 ) and processed for small RNA - sequencing . The log2 - fold induction levels for tRFs derived from distinct tRNA isoacceptor is plotted .

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Figure 1(A) Heatmap of tRNA profiling of MCF10A cells at 8 and 24 hours post exposure to oxidative stress (200μM H2O2). Biological triplicate data is depicted at each time point relative to control cells.(B) MCF10A cells were exposed to oxidative stress (200μM H2O2) and processed for small RNA-sequencing. The log2-fold induction levels for tRFs derived from distinct tRNA isoacceptor is plotted.


Figure 2 ( A ) A northern blot depicting a time course experiment ranging from five minutes to 24 hours of MCF10A cells in response to oxidative stress . A single probe complementary to pre - tRNATyrGUA , mature tRNATyrGUA , and tRFTyrGUA expression was 32P - labeled and used for detection . ( B ) Quantification of pre - tRNATyrGUA ( left ) and mature tRNATyrGUA levels ( right ) by northern blot analysis from multiple independent experiments ( normalized to U6 levels ) are shown ( n = 6 ) . ( C ) MCF10A cells were exposed to oxidative stress ( 200μM H2O2 ) once daily for five continuous days . ( D ) Quantification of mature tRNATyrGUA bands by northern blot after cells were treated once daily for five continuous days ( normalized to U6 ) from multiple independent experiments ( n = 12 ) . ( E ) Quantification of northern blot analysis for pre - tRNATyrGUA ( left ) and tRNATyrGUA ( right ) after one hour and 24 hours respectively in HBEC30 cells upon exposure to oxidative stress ( 200μM H2O2 ) as in ( A ) ( n = 6 ) .

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Figure 2(A) A northern blot depicting a time course experiment ranging from five minutes to 24 hours of MCF10A cells in response to oxidative stress. A single probe complementary to pre-tRNATyrGUA, mature tRNATyrGUA, and tRFTyrGUA expression was 32P-labeled and used for detection. (B) Quantification of pre-tRNATyrGUA (left) and mature tRNATyrGUA levels (right) by northern blot analysis from multiple independent experiments (normalized to U6 levels) are shown (n=6). (C) MCF10A cells were exposed to oxidative stress (200μM H2O2) once daily for five continuous days. (D) Quantification of mature tRNATyrGUA bands by northern blot after cells were treated once daily for five continuous days (normalized to U6) from multiple independent experiments (n=12). (E) Quantification of northern blot analysis for pre-tRNATyrGUA (left) and tRNATyrGUA (right) after one hour and 24 hours respectively in HBEC30 cells upon exposure to oxidative stress (200μM H2O2) as in (A) (n=6).