( 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 ) .

(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).


( 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 .

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

(a) 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 .

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

(c) 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 .

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

(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 .

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

(e,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 .

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

(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 .

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

(h) 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(i) 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.


( 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 .

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

(a) 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 .

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

(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 .

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

(d) 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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.


( 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 .

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

(a,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 .

(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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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.


( 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 .

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

(a) 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 .

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

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

(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.


( 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 ) .

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

(a) 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 ) .

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

(c) 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 .

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

(d-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 .

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

(h-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 ) .

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

(l) 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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).


B - 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 .

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

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

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 ###,***).


A . AhR Protein levels in 501Mel CTR cells and those transduced with sgRNA targeting AhR ( # 1 , # 2 ) were analyzed by western blotting .

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

A. 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 * * * .

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

C. 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 .

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

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

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.


A . 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 .

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A. 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 .

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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 ) .

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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 ) .

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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 .

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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.


B . 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 * * * .

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B. 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 .

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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 ) .

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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 .

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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.


B . 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 * * .

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B. 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 .

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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.


Week 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 ) .

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Week 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 .

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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.


Sample 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 ) .

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Sample 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 .

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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 .


Week 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 =

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Week 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 = 100µm


IF 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 .

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IF 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.


RNA 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 .

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RNA 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 ) ] .

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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 .

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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.


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 . 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 .

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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.   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 .

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

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.


Week 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 .

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

Week 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 .

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

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

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


A 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 ) .

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

A 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 .

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

B The 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 .

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

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 ) .

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

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

E,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.


A 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 .

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

A 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 .

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

B The 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 .

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

C 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 .

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

D 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E, 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.


A 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 .

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

A RPE 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 ) .

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

B The 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 .

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

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 ) .

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 .

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.


A - 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 .

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

A-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 O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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.


A 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 .

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

A 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 ) .

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

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 ) .

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

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

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.


A , 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 ) .

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

A,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 .

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

C 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 .

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

D-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 .

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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.


A 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 ) .

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A 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 .

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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 ) .

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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).


A 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 .

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A 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 .

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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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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.


( 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 .

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(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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(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.


( 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 ) .

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(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 ) .

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(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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(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).


( A ) Growth curves of MCF10A cells exposed to oxidative stress ( 200μM H2O2 ) relative to control cells ( n = 3 ) . Two - way ANOVA was used to test for significance .

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(A) Growth curves of MCF10A cells exposed to oxidative stress (200μM H2O2) relative to control cells (n=3). Two-way ANOVA was used to test for significance.


( B ) Northern blot of MCF10A cells expressing a control short - hairpin RNA or a hairpin targeting tRNATyrGUA .

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(B) Northern blot of MCF10A cells expressing a control short-hairpin RNA or a hairpin targeting tRNATyrGUA.


( C ) Western blot of MCF10A expressing a control short - hairpin RNA or a hairpin targeting the tyrosine - tRNA synthetase , YARS ( red arrow ) . HSC70 was used as a loading control .

(C) Western blot of MCF10A expressing a control short-hairpin RNA or a hairpin targeting the tyrosine-tRNA synthetase, YARS (red arrow). HSC70 was used as a loading control.


( D ) Growth curves of MCF10A cells expressing RNAi targeting mature tRNATyrGUA or YARS relative to cells expressing a control hairpin ( n = 3 ) . Two - way ANOVA was used to test for significance .

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(D) Growth curves of MCF10A cells expressing RNAi targeting mature tRNATyrGUA or YARS relative to cells expressing a control hairpin (n=3). Two-way ANOVA was used to test for significance.


( E ) Cell growth of MCF10A cells transiently transfected with a tRNATyrGUA overexpression vector relative to an empty control vector ( n = 3 ) . A one - tailed Mann - Whitney test was used to test for significance at day 3 .

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(E) Cell growth of MCF10A cells transiently transfected with a tRNATyrGUA overexpression vector relative to an empty control vector (n=3). A one-tailed Mann-Whitney test was used to test for significance at day 3.


( A ) Cells depleted of tRNATyrGUA or YARS were processed for label free quantitation by mass spectrometry to identify proteins that were reduced by a log2 - fold change of 0 . 5 or more . This set was overlapped with proteins containing a higher than median abundance of Tyr codon content to identify candidate mediators of the pleiotropic effects of tRNATyrGUA depletion .

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(A) Cells depleted of tRNATyrGUA or YARS were processed for label free quantitation by mass spectrometry to identify proteins that were reduced by a log2-fold change of 0.5 or more. This set was overlapped with proteins containing a higher than median abundance of Tyr codon content to identify candidate mediators of the pleiotropic effects of tRNATyrGUA depletion.


( C ) Quantitative western blot validation depicting abundances of protein targets ( EPCAM , SCD , and USP3 ) identified from ( A ) . HSC70 was used as a loading control and is not modulated upon molecular perturbation of tRNATyrGUA . ( D ) Quantification of western blot analysis in ( C ) ( n = 4 ) . A one - tailed Mann - Whitney test was used to test for statistical significance .

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(C) Quantitative western blot validation depicting abundances of protein targets (EPCAM, SCD, and USP3) identified from (A). HSC70 was used as a loading control and is not modulated upon molecular perturbation of tRNATyrGUA. (D) Quantification of western blot analysis in (C) (n=4). A one-tailed Mann-Whitney test was used to test for statistical significance.


( E ) Growth curves for MCF10A cells were transfected with either control siRNA or two independent siRNA targeting EPCAM , SCD , or USP3 . Note that the control cell growth curve is the same in all graphs and were plotted separately for clarity and does not represent independent experiments . Two - way ANOVA was used to test for significance ( n = 3 ) .

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(E) Growth curves for MCF10A cells were transfected with either control siRNA or two independent siRNA targeting EPCAM, SCD, or USP3. Note that the control cell growth curve is the same in all graphs and were plotted separately for clarity and does not represent independent experiments. Two-way ANOVA was used to test for significance (n=3).


( A ) Quantitative western blot EPCAM , SCD , and USP3 in MCF10A cells 24 hours after treatment with H2O2 ( 200μM ) . HSC70 was used as a loading control . ( B ) Quantification of western results in ( A ) ( n = 9 ) . A one - tailed Mann - Whitney test was used to test for statistical significance .

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(A) Quantitative western blot EPCAM, SCD, and USP3 in MCF10A cells 24 hours after treatment with H2O2 (200μM). HSC70 was used as a loading control. (B) Quantification of western results in (A) (n=9). A one-tailed Mann-Whitney test was used to test for statistical significance.


( D ) Quantitative western blot for the Myc - tag and Luciferase ( top ) with normalized fluorescent intensities ( bottom ) are shown ( n = 3 ) .

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(D) Quantitative western blot for the Myc-tag and Luciferase (top) with normalized fluorescent intensities (bottom) are shown (n=3).


( E ) Ribosome occupancy of 20 - 22nt RPFs in tRNATyrGUA - depleted cells compared to control cells reveal greater occupancy at both tyrosine codons in tRNATyrGUA - depleted cells using a Wilcoxon test .

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(E) Ribosome occupancy of 20-22nt RPFs in tRNATyrGUA-depleted cells compared to control cells reveal greater occupancy at both tyrosine codons in tRNATyrGUA-depleted cells using a Wilcoxon test.


( A - B ) Quantification of tRFTyrGUA induction in response to oxidative stress as a function of time in MCF10A ( A ) ( n = 4 ) and in HBEC30 ( B ) ( n = 6 ) with mean ± s . e . m shown for each cell line .

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(A-B) Quantification of tRFTyrGUA induction in response to oxidative stress as a function of time in MCF10A (A) (n=4) and in HBEC30 (B) (n=6) with mean ± s.e.m shown for each cell line.


( C ) Volcano plot of mass spectrometry results from a synthetic 5 ' - biotinylated tRFTyrGUA co - precipitation experiment with cell lysate . Log2 fold enrichment values of proteins identified from tRFTyrGUA relative to scrambled tRF control samples .

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(C) Volcano plot of mass spectrometry results from a synthetic 5'-biotinylated tRFTyrGUA co-precipitation experiment with cell lysate. Log2 fold enrichment values of proteins identified from tRFTyrGUA relative to scrambled tRF control samples.


( D ) Western blot validation of mass spectrometry results for three of the top hits in ( A ) , showing co - precipitation of endogenous proteins with transfected tRFTyrGUA relative to a scrambled control sequence ( Scr ) .

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(D) Western blot validation of mass spectrometry results for three of the top hits in (A), showing co-precipitation of endogenous proteins with transfected tRFTyrGUA relative to a scrambled control sequence (Scr).


( B ) IGV plots from an hnRNPA1 HITS - CLIP ( Huelga et al . , 2012 ) reveals interactions with tRFTyrGUA .

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(B) IGV plots from an hnRNPA1 HITS-CLIP (Huelga et al., 2012) reveals interactions with tRFTyrGUA.


( C ) IGV plots representing SSB interacting with the tRFTyrGUA in samples that were treated with low levels of RNase digestion . SSB bound tRFTyrGUA reads mapped to multiple loci encoding tRNATyrGUA . ( D ) Similar to the IGV plots shown in ( C ) , but depicting SSB interactions with tRFTyrGUA loci in samples without RNase digestion .

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(C) IGV plots representing SSB interacting with the tRFTyrGUA in samples that were treated with low levels of RNase digestion. SSB bound tRFTyrGUA reads mapped to multiple loci encoding tRNATyrGUA. (D) Similar to the IGV plots shown in (C), but depicting SSB interactions with tRFTyrGUA loci in samples without RNase digestion.


( E ) A cumulative distribution in control and hnRNPA1 depleted cells of the stability levels for mRNA transcripts with 3 ' UTR hnRNPA1 CLIP binding ( Huelga et al . , 2012 ) . Transfection of tRFTyrGUA led to a significant right - shift in the expression levels of 3 ' UTR bound hnRNPA1 transcripts . Statistical significance was measured using the Kolmogorov - Smirnov test . ( F ) Cumulative distribution as in ( E ) . Transfection of locked nucleic acid against tRFTyrGUA and treatment with 200µM H2O2 led to a significant left - shift in mRNA stability of 3 ' UTR bound hnRNPA1 transcripts . Statistical significance was assessed using the Kolmogorov - Smirnov test .

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(E) A cumulative distribution in control and hnRNPA1 depleted cells of the stability levels for mRNA transcripts with 3' UTR hnRNPA1 CLIP binding (Huelga et al., 2012). Transfection of tRFTyrGUA led to a significant right-shift in the expression levels of 3' UTR bound hnRNPA1 transcripts. Statistical significance was measured using the Kolmogorov-Smirnov test. (F) Cumulative distribution as in (E). Transfection of locked nucleic acid against tRFTyrGUA and treatment with 200µM H2O2 led to a significant left-shift in mRNA stability of 3'UTR bound hnRNPA1 transcripts. Statistical significance was assessed using the Kolmogorov-Smirnov test.


( A ) Expression levels of NAT4 analyzed by qRT - PCR using total RNA extracted from a wild - type ( BY4741 ) and Pste5 - NAT4 strain grown in 2 % ( NCR ) , or 0 . 1 % glucose ( CR ) . NAT4 levels were normalized to TAF10 , whose expression remains unchanged . Error bars , SEM ( Standard Error of the Mean ) of 3 independent experiments . * * p ≤ 0 . 01 ; calculated by unpaired two - tailed Student ' s t - test .

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(A) Expression levels of NAT4 analyzed by qRT-PCR using total RNA extracted from a wild-type (BY4741) and Pste5-NAT4 strain grown in 2% (NCR), or 0.1% glucose (CR). NAT4 levels were normalized to TAF10, whose expression remains unchanged. Error bars, SEM (Standard Error of the Mean) of 3 independent experiments. ** p ≤ 0.01; calculated by unpaired two-tailed Student's t-test.


( B ) ChIP analysis performed in BY4741 wild - type , nat4Δ and Pste5 - NAT4 strains grown in the same conditions as in ( A ) . Chromatin was immunoprecipitated using an antibody against N - acH4 and analyzed by qRT - PCR using primers for the different rDNA regions . Enrichment was normalized to Histone H4 levels . Error bars , SEM of 3 independent experiments . Statistical significance was determined by unpaired two - tailed Student ' s t test : * p ≤ 0 . 05 ; * * p ≤ 0 . 01 compared values to wild - type NCR conditions ; arrowhead p ≤ 0 . 05 compared values to wild - type CR

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(B) ChIP analysis performed in BY4741 wild-type, nat4Δ and Pste5-NAT4 strains grown in the same conditions as in (A). Chromatin was immunoprecipitated using an antibody against N-acH4 and analyzed by qRT-PCR using primers for the different rDNA regions. Enrichment was normalized to Histone H4 levels. Error bars, SEM of 3 independent experiments. Statistical significance was determined by unpaired two-tailed Student's t test: * p ≤ 0.05; ** p ≤ 0.01 compared values to wild-type NCR conditions; arrowhead p ≤ 0.05 compared values to wild-type CR conditions


( C ) Replicative lifespan ( RLS ) for BY4741 wild - type and nat4Δ strains . Values in parenthesis ( here and hereafter ) indicate mean lifespan . Statistical significance ( here and hereafter ) was determined by one - way ANOVA test : * p ≤ 0 . 05 ; * * p ≤ 0 . 01 ; * * * p ≤ 0 . 001 ; * * * * p ≤ 0 . 0001 .

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(C) Replicative lifespan (RLS) for BY4741 wild-type and nat4Δ strains. Values in parenthesis (here and hereafter) indicate mean lifespan. Statistical significance (here and hereafter) was determined by one-way ANOVA test: * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001.


( D ) Mean RLS for wild - type and nat4 ∆ strains using different genetic backgrounds . Values outside of parenthesis indicate mean lifespan , while values in parenthesis indicate the number of cells examined .

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(D) Mean RLS for wild-type and nat4∆ strains using different genetic backgrounds. Values outside of parenthesis indicate mean lifespan, while values in parenthesis indicate the number of cells examined.


( E ) Mean RLS for BY4741 wild - type and nat4Δ strains under NCR and CR conditions .

(E) Mean RLS for BY4741 wild-type and nat4Δ strains under NCR and CR conditions.


( F ) Mean RLS for BY4741 wild - type and Pste5 - NAT4 strains under NCR and CR conditions .

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(F) Mean RLS for BY4741 wild-type and Pste5-NAT4 strains under NCR and CR conditions.