Age - dependent protein levels of MURF1 ( L ) , and ATROGIN1 ( N ) estimated by band densitometry normalized by GAPDH , in COX10 KO expressed relative to same age CTL set at 1 . Representative western blot of muscle lysates from 100 and 170 - 200 days old mice separated by denaturing SDS - PAGE and probed for ( M ) , ATROGIN1 ( O ) , and GAPDH In panels data are presented as Mean ± SD . COX10 KO ( n = 4 ) , CTL ( n = 4 ) . * , p < 0 . 05 COX10 KO vs . same age CTL .

B-EXP_ASSAY O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O B-EXP_ASSAY O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-TISSUE O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O

Age-dependent protein levels of MURF1 (L), and ATROGIN1 (N) estimated by band densitometry normalized by GAPDH, in COX10 KO expressed relative to same age CTL set at 1. Representative western blot of muscle lysates from 100 and 170-200 days old mice separated by denaturing SDS-PAGE and probed for (M), ATROGIN1 (O), and GAPDH In panels data are presented as Mean ± SD. COX10 KO (n=4), CTL (n=4). *, p<0.05 COX10 KO vs. same age CTL.


( P ) Ph - 4EBP1 / 4E - BP1 ratio and SESTRIN 2 protein levels normalized by α - actin , estimated by band densitometry , in MERRF muscle expressed relative to CTL muscle set at 1 . ( Q ) Representative western blot of MERRF and CTL muscle lysates separated by denaturing SDS - PAGE and probed for Ph - 4EBP1 , 4EBP1 , SESTRIN 2 , and α - actin . In panel P , data are presented as Mean ± SD . MERRF ( n = 4 ) , CTL ( n = 4 ) . * , p < 0 . 05 MERRF vs . CTL . Statistically significant differences between the two groups for all panels were estimated by unpaired two - tailed Student ' s test .

O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-EXP_ASSAY O O B-DISEASE B-TISSUE O O O O B-TISSUE O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-DISEASE O O B-TISSUE O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O B-DISEASE O O O O O O O O O O O O O O O O O O O O B-DISEASE O O O O O O O O O O O O O O O O O O O O O O O O O O

(P) Ph-4EBP1/4E-BP1 ratio and SESTRIN 2 protein levels normalized by α-actin, estimated by band densitometry, in MERRF muscle expressed relative to CTL muscle set at 1. (Q) Representative western blot of MERRF and CTL muscle lysates separated by denaturing SDS-PAGE and probed for Ph-4EBP1, 4EBP1, SESTRIN 2, and α-actin. In panel P, data are presented as Mean ± SD. MERRF (n=4), CTL (n=4). *, p<0.05 MERRF vs. CTL. Statistically significant differences between the two groups for all panels were estimated by unpaired two-tailed Student's test.


Age - dependent levels of ATF4 ( B ) , MTHFD2 ( D ) estimated by band densitometry normalized by GAPDH , in COX10 KO expressed relative to same age CTL set at 1 . Representative western blot of muscle lysates from 200 days old mice separated by denaturing SDS - PAGE and probed for ATF4 ( C ) , MTHFD2 ( E ) and GAPDH In panels data are presented as Mean ± SD . * , p < 0 . 05 COX10 KO vs . same age CTL . For each age , COX10 KO ( n = 4 ) , CTL ( n - 4 ) .

O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O B-EXP_ASSAY O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-TISSUE O O O O O B-ORGANISM O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O

Age-dependent levels of ATF4 (B), MTHFD2 (D) estimated by band densitometry normalized by GAPDH, in COX10 KO expressed relative to same age CTL set at 1. Representative western blot of muscle lysates from 200 days old mice separated by denaturing SDS-PAGE and probed for ATF4 (C), MTHFD2 (E) and GAPDH In panels data are presented as Mean ± SD. *, p<0.05 COX10 KO vs. same age CTL. For each age, COX10 KO (n=4), CTL (n-4).


Age - dependent levels of ASNS ( F ) , and HSP60 ( H ) , estimated by band densitometry normalized by GAPDH , in COX10 KO expressed relative to same age CTL set at 1 . Representative western blot of muscle lysates from 200 days old mice separated by denaturing SDS - PAGE and probed for ASNS ( G ) , and HSP60 ( I ) and GAPDH In panels data are presented as Mean ± SD . * , p < 0 . 05 COX10 KO vs . same age CTL . For each age , COX10 KO ( n = 4 ) , CTL ( n - 4 ) .

O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O B-EXP_ASSAY O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-TISSUE O O O O O B-ORGANISM O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O

Age-dependent levels of ASNS (F), and HSP60 (H), estimated by band densitometry normalized by GAPDH, in COX10 KO expressed relative to same age CTL set at 1. Representative western blot of muscle lysates from 200 days old mice separated by denaturing SDS-PAGE and probed for ASNS (G), and HSP60 (I) and GAPDH In panels data are presented as Mean ± SD. *, p<0.05 COX10 KO vs. same age CTL. For each age, COX10 KO (n=4), CTL (n-4).


( N ) Levels of metabolites of trans - sulfuration pathway by LC - MS analysis in MERRF muscle and plasma expressed relative to CTL muscle and plasma . α - HB , α - hydroxybutyrate ; GSH , glutathione . In panel N , data are presented as Mean ± SEM . Muscle : MERRF ( n = 10 ) , CTL ( n = 15 ) . Plasma : MERRF ( n = 9 ) , CTL ( n = 25 ) . * p < 0 . 05 MERRF vs . CTL . Statistically significant differences between the two groups for all panels were estimated by unpaired two - tailed Student ' s test .

O O O O O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O B-DISEASE B-TISSUE O B-TISSUE O O O O B-TISSUE O B-TISSUE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O B-TISSUE O B-DISEASE O O O O O O O O O O O O O B-TISSUE O B-DISEASE O O O O O O O O O O O O O O O O O O O B-DISEASE O O O O O O O O O O O O O O O O O O O O O O O O O O

(N) Levels of metabolites of trans-sulfuration pathway by LC-MS analysis in MERRF muscle and plasma expressed relative to CTL muscle and plasma. α-HB, α-hydroxybutyrate; GSH, glutathione. In panel N, data are presented as Mean ± SEM. Muscle: MERRF (n=10), CTL (n=15). Plasma: MERRF (n=9), CTL (n=25). * p<0.05 MERRF vs. CTL. Statistically significant differences between the two groups for all panels were estimated by unpaired two-tailed Student's test.


( D - I ) Age - dependent levels of HADH ( D ) , ACOT2 ( F ) , and ACOT9 ( H ) , estimated by band densitometry normalized by GAPDH , in COX10 KO expressed relative to same age CTL set at 1 . Representative western blot of muscle lysates from 200 days old mice separated by denaturing SDS - PAGE and probed for HADH ( E ) , ACOT2 ( G ) , ACOT9 ( I ) , and GAPDH ( E , G , I ) . In panels , data are presented as Mean ± SD . COX10 KO ( n = 4 ) , CTL ( n = 4 ) . * , p < 0 . 05 COX10 KO vs . same age CTL .

O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O B-EXP_ASSAY O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-TISSUE O O O O O B-ORGANISM O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O

(D-I) Age-dependent levels of HADH (D), ACOT2 (F), and ACOT9 (H), estimated by band densitometry normalized by GAPDH, in COX10 KO expressed relative to same age CTL set at 1. Representative western blot of muscle lysates from 200 days old mice separated by denaturing SDS-PAGE and probed for HADH (E), ACOT2 (G), ACOT9 (I), and GAPDH (E, G, I). In panels , data are presented as Mean ± SD. COX10 KO (n=4), CTL (n=4). *, p<0.05 COX10 KO vs. same age CTL.


( K , L ) Levels of acylcarnitine , 3 - OH acylcarnitine , fatty acid and 3 - OH fatty acid in MERRF muscle ( K ) and plasma ( L ) by LC - MS analysis , expressed relative to CTL . In panels data by LC - MS analysis are presented as Mean ± SEM . In panels A and C , 50 , 100 , 200d : COX10 KO ( n = 6 ) , CTL ( n = 6 ) . * , p < 0 . 05 COX10 KO vs . same age CTL . In panels K and L , muscle : MERRF ( n = 10 ) , CTL ( n = 15 ) ; plasma : MERRF ( n = 9 ) , CTL ( n = 25 ) . * , p < 0 . 05 MERRF vs . CTL .

O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-DISEASE B-TISSUE O O O O B-TISSUE O O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O B-TISSUE O B-DISEASE O O O O O O O O O O O O O B-TISSUE O B-DISEASE O O O O O O O O O O O O O O O O O O O O B-DISEASE O O O O

(K, L) Levels of acylcarnitine, 3-OH acylcarnitine, fatty acid and 3-OH fatty acid in MERRF muscle (K) and plasma (L) by LC-MS analysis, expressed relative to CTL. In panels data by LC-MS analysis are presented as Mean ± SEM. In panels A and C, 50, 100, 200d: COX10 KO (n=6), CTL (n=6). *, p<0.05 COX10 KO vs. same age CTL. In panels K and L, muscle: MERRF (n=10), CTL (n=15); plasma: MERRF (n=9), CTL (n=25). *, p<0.05 MERRF vs. CTL.


Age - dependent muscle protein levels of P5CS ( B ) , PYCR1 ( D ) estimated by band densitometry normalized by GAPDH , in COX10 KO expressed relative to same age CTL set at 1 . Data are presented as Mean ± SD . * , p < 0 . 05 COX10 KO vs . same age CTL . Representative western blots of muscle lysates from 170 days old mice separated by denaturing SDS - PAGE and probed for P5CS ( C ) , PYCR1 ( E ) GAPDH In panels data are normalized to same age CTL set to 1 . Data are presented as Mean ± SD . 50 , 100 , 200d : COX10 KO ( n = 4 ) , CTL ( n = 4 ) . * , p < 0 . 05 COX10 KO vs . CTL .

O O O B-TISSUE O O O B-GENEPROD O O O O B-GENEPROD O O O O O O B-EXP_ASSAY O O B-GENEPROD O O B-GENEPROD O O 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-GENEPROD O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-TISSUE O O O O O B-ORGANISM O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O O O 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-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O

Age-dependent muscle protein levels of P5CS (B), PYCR1 (D) estimated by band densitometry normalized by GAPDH, in COX10 KO expressed relative to same age CTL set at 1. Data are presented as Mean ± SD. *, p<0.05 COX10 KO vs. same age CTL. Representative western blots of muscle lysates from 170 days old mice separated by denaturing SDS-PAGE and probed for P5CS (C), PYCR1 (E) GAPDH In panels data are normalized to same age CTL set to 1. Data are presented as Mean ± SD. 50, 100, 200d: COX10 KO (n=4), CTL (n=4). *, p<0.05 COX10 KO vs. CTL.


Age - dependent muscle protein levels of PYCR2 ( F ) OAT ( H ) , estimated by band densitometry normalized by GAPDH , in COX10 KO expressed relative to same age CTL set at 1 . Data are presented as Mean ± SD . * , p < 0 . 05 COX10 KO vs . same age CTL . Representative western blots of muscle lysates from 170 days old mice separated by denaturing SDS - PAGE and probed for , PYCR2 ( G ) , OAT ( I ) , and GAPDH : In panels data are normalized to same age CTL set to 1 . Data are presented as Mean ± SD . 50 , 100 , 200d : COX10 KO ( n = 4 ) , CTL ( n = 4 ) . * , p < 0 . 05 COX10 KO vs . CTL .

O O O B-TISSUE O O O B-GENEPROD O O O B-GENEPROD O O O O O O O B-EXP_ASSAY O O B-GENEPROD O O B-GENEPROD O O 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-GENEPROD O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-TISSUE O O O O O B-ORGANISM O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O B-GENEPROD O O O O 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-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O

Age-dependent muscle protein levels of PYCR2 (F) OAT (H), estimated by band densitometry normalized by GAPDH, in COX10 KO expressed relative to same age CTL set at 1. Data are presented as Mean ± SD. *, p<0.05 COX10 KO vs. same age CTL. Representative western blots of muscle lysates from 170 days old mice separated by denaturing SDS-PAGE and probed for , PYCR2 (G), OAT (I), and GAPDH : In panels data are normalized to same age CTL set to 1. Data are presented as Mean ± SD. 50, 100, 200d: COX10 KO (n=4), CTL (n=4). *, p<0.05 COX10 KO vs. CTL.


( M ) MERRF plasma levels of 4 - hydroxyl - L - proline by LC - MS analysis , expressed relative to CTL value set at 1 .

O O O B-DISEASE B-TISSUE O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O O

(M) MERRF plasma levels of 4-hydroxyl-L-proline by LC-MS analysis, expressed relative to CTL value set at 1.


( N ) PYCR1 protein levels , estimated by band densitometry normalized by GAPDH , in MERRF muscle expressed relative to CTL muscle . In panel N , data are presented as Mean ± SD . MERRF ( n = 4 ) , CTL ( n = 4 ) . * , p < 0 . 05 MERRF vs . CTL . Statistically significant differences between the two groups for all panels were estimated by unpaired two - tailed Student ' s test .

O O O B-GENEPROD O O O O O O B-EXP_ASSAY O O B-GENEPROD O O B-DISEASE B-TISSUE O O O O B-TISSUE O O O O O O O O O O O O O B-DISEASE O O O O O O O O O O O O O O O O O O O O B-DISEASE O O O O O O O O O O O O O O O O O O O O O O O O O O

(N) PYCR1 protein levels, estimated by band densitometry normalized by GAPDH, in MERRF muscle expressed relative to CTL muscle. In panel N, data are presented as Mean ± SD. MERRF (n=4), CTL (n=4). *, p<0.05 MERRF vs. CTL. Statistically significant differences between the two groups for all panels were estimated by unpaired two-tailed Student's test.


( A - C ) Plasma levels of Leptin ( A ) , soluble Leptin Receptor ( LepR ) , ( B ) and Free Leptin Index ( C ) by ELISA , in 200 days old CTL and COX10 KO mice . In panels A - C , data are presented as Mean ± SD . COX10 KO ( n = 5 ) , CTL ( n = 5 ) . * , p < 0 . 05 COX10 KO vs . CTL .

O O O O O B-TISSUE O O B-GENEPROD O O O O O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O O O O O B-EXP_ASSAY O O O O O O O B-GENEPROD O B-ORGANISM O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O

(A-C) Plasma levels of Leptin (A), soluble Leptin Receptor (LepR), (B) and Free Leptin Index (C) by ELISA, in 200 days old CTL and COX10 KO mice. In panels A-C, data are presented as Mean ± SD. COX10 KO (n=5), CTL (n=5). *, p<0.05 COX10 KO vs. CTL.


( G ) Levels of glucocorticoids in MERRF plasma by LC - MS analysis , expressed relative to CTL value set at 1 . In panels G data are presented as Mean ± SEM . MERRF ( n = 9 ) , CTL ( n = 25 ) .

O O O O O B-SMALL_MOLECULE O B-DISEASE B-TISSUE O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O O O 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) Levels of glucocorticoids in MERRF plasma by LC-MS analysis, expressed relative to CTL value set at 1. In panels G data are presented as Mean ± SEM. MERRF (n=9), CTL (n=25).


( N ) Age - dependent grip strength of COX10 KO mice treated with RU - 486 and vehicle . The black arrow indicates the beginning of the treatment at 43 days . In panel data are presented as Mean ± SEM . Panel N : KO + RU486 ( n = 6 ) , KO ( n = 4 ) .

O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-ORGANISM O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O 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-SMALL_MOLECULE O O O O O O O O O O O O O

(N) Age-dependent grip strength of COX10 KO mice treated with RU-486 and vehicle. The black arrow indicates the beginning of the treatment at 43 days. In panel data are presented as Mean ± SEM. Panel N: KO+RU486 (n=6), KO (n=4).


( P ) Visceral fat deposits of COX10 KO mice treated with RU - 486 and vehicle at 100 days of age . In panel P , data are presented as Mean ± SD . KO + RU486 ( n = 6 ) , KO ( n = 6 ) . * , p < 0 . 05 KO + RU486 vs . KO . Statistically significant differences between the two groups for all panels were estimated by unpaired two - tailed Student ' s test .

O O O B-TISSUE I-TISSUE O O B-GENEPROD O B-ORGANISM O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O

(P) Visceral fat deposits of COX10 KO mice treated with RU-486 and vehicle at 100 days of age. In panel P, data are presented as Mean ± SD. KO+RU486 (n=6), KO (n=6). *, p<0.05 KO+RU486 vs. KO. Statistically significant differences between the two groups for all panels were estimated by unpaired two-tailed Student's test.


( A ) GAPC enzymatic activity was measured in wild - type crude extracts treated with H2O ( mock ) , and 100 µM of Tyr - Asp , Tyr , Asp , and Ile - Glu . ( B - D ) The enzymatic activities of GAPC ( B ) , GAPA / B ( C ) , and GAPN ( D ) were measured in the wild type and double k . o . mutant gapc1gapc2 treated with H2O ( mock ) , and 100 µM of Tyr - Asp . Data information : Data are mean ± SEM of n = 4 ( A - D ; four technical replicates ) , significance was assessed using unpaired two - tailed Student t - test .

O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O 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) GAPC enzymatic activity was measured in wild-type crude extracts treated with H2O (mock), and 100 µM of Tyr-Asp, Tyr, Asp, and Ile-Glu. (B-D) The enzymatic activities of GAPC (B), GAPA/B (C), and GAPN (D) were measured in the wild type and double k.o. mutant gapc1gapc2 treated with H2O (mock), and 100 µM of Tyr-Asp. Data information: Data are mean ± SEM of n=4 (A-D; four technical replicates) , significance was assessed using unpaired two-tailed Student t-test.


( A ) Fresh weight quantification of Arabidopsis and tobacco seedlings . Seedlings were pretreated with mock ( water ) , Tyr - Asp ( 100 μM ) , Tyr and Asp ( 100 μM ) , Ser - Leu ( 100 μM ) , or Gly - Pro ( 100 μM ) for 1 h prior an oxidative stress ( catechin or H2O2 ) . Each treatment was compared to its respective control , corresponding to plants grown in one 24 - well plate ( represented by the adjoined bars ) . Data information : Data are mean ± SEM of n = 10 - 12 ( seedlings ) . Unpaired two - tailed Student t - test was performed to compare treatments with control . Scale bar : 10 cm .

O O O O B-EXP_ASSAY O O B-ORGANISM O B-ORGANISM O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Fresh weight quantification of Arabidopsis and tobacco seedlings. Seedlings were pretreated with mock (water), Tyr-Asp (100 μM), Tyr and Asp (100 μM), Ser-Leu (100 μM), or Gly-Pro (100 μM) for 1 h prior an oxidative stress (catechin or H2O2). Each treatment was compared to its respective control, corresponding to plants grown in one 24-well plate (represented by the adjoined bars). Data information: Data are mean ± SEM of n=10-12 (seedlings). Unpaired two-tailed Student t-test was performed to compare treatments with control. Scale bar: 10 cm.


( B ) Catechin - ( left upper panel ) and ( catechin - ) Tyr - Asp - treated tobacco plants ( right upper panel ) grown for four days in liquid media . H2O2 - ( left upper panel ) and ( H2O2 - ) Tyr - Asp - treated tobacco plants ( right upper panel ) grown for two days in liquid media . All plants were 2 - weeks - old before starting the stress treatment . Data information : Data are mean ± SEM of n = 10 - 12 ( seedlings ) .

O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-ORGANISM O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O 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) Catechin- (left upper panel) and (catechin-) Tyr-Asp-treated tobacco plants (right upper panel) grown for four days in liquid media. H2O2- (left upper panel) and (H2O2-) Tyr-Asp-treated tobacco plants (right upper panel) grown for two days in liquid media. All plants were 2-weeks-old before starting the stress treatment. Data information: Data are mean ± SEM of n=10-12 (seedlings).


( A - C ) Fresh weight measurements of Arabidopsis wild - type and gapc1 / gapc2 double k . o . plants subjected to the oxidative stress ( e . g . catechin ) and Tyr - Asp treatments . Each treatment was compared to its respective control , corresponding to plants grown in one 24 - well plate ( represented by the adjoined bars ) . ( D - F ) Representative Arabidopsis wild type and gapc1 / gapc2 double k . o . seedlings from the different treatments . All plants were 10 - day - old at the stress onset . The seedlings were exposed to catechin for three days . Data information : Data are mean ± SEM of n = 10 - 12 ( seedlings ) . Unpaired two - tailed Student t - test was performed to compare treatments with control . Scale bar : 10 cm .

O O O O O O B-EXP_ASSAY O O B-ORGANISM O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O 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-ORGANISM O O O B-GENEPROD O B-GENEPROD O O O O O 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-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O 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) Fresh weight measurements of Arabidopsis wild-type and gapc1/gapc2 double k.o. plants subjected to the oxidative stress (e.g. catechin) and Tyr-Asp treatments. Each treatment was compared to its respective control, corresponding to plants grown in one 24-well plate (represented by the adjoined bars). (D-F) Representative Arabidopsis wild type and gapc1/gapc2 double k.o. seedlings from the different treatments. All plants were 10-day-old at the stress onset. The seedlings were exposed to catechin for three days. Data information: Data are mean ± SEM of n=10-12 (seedlings). Unpaired two-tailed Student t-test was performed to compare treatments with control. Scale bar: 10 cm.


( C ) PEPCK activity was measured in a protein lysate prepared from 3 - week - old Arabidopsis leaves in absence ( control , C ) or presence of 500 µM of the different dipeptides . Measurements were performed in the carboxylation direction , as described in the Methods section . Data are mean ± SE of 4 independent lysate preparations . Unpaired two - tailed Student t - tests were performed to compare dipeptide treatments with the control .

O O O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O B-ORGANISM B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) PEPCK activity was measured in a protein lysate prepared from 3-week-old Arabidopsis leaves in absence (control, C) or presence of 500 µM of the different dipeptides. Measurements were performed in the carboxylation direction, as described in the Methods section. Data are mean ± SE of 4 independent lysate preparations. Unpaired two-tailed Student t-tests were performed to compare dipeptide treatments with the control.


( A ) Mettl14 cKO leads to perinatal lethality . Newborn cKO pups are smaller with tight and shiny skin .

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(A) Mettl14 cKO leads to perinatal lethality. Newborn cKO pups are smaller with tight and shiny skin.


( B ) Immunohistochemistry confirms loss of Mettl14 expression in skin in cKO animals .

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(B) Immunohistochemistry confirms loss of Mettl14 expression in skin in cKO animals.


( C ) H / E staining of newborn skin sections from WT and Mettl14 cKO mice .

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(C) H/E staining of newborn skin sections from WT and Mettl14 cKO mice.


( D ) Expression of p63 in skin epidermis was examined by immunofluorescence staining in WT and cKO mice . ( E ) Number of p63 - positive cells in WT and cKO skin was quantified and shown as box and whisker plots . The plot indicates the mean ( solid diamond within the box ) , 25th percentile ( bottom line of the box ) , median ( middle line of the box ) , 75th percentile ( top line of the box ) , 5th and 95th percentile ( whiskers ) , 1st and 99th percentile ( solid triangles ) and minimum and maximum measurements ( solid squares ) . n = 6 ( biological repeats ) , P < 0 . 05 ( Student ' s t - test ) .

O O O B-EXP_ASSAY O B-GENEPROD O B-TISSUE I-TISSUE O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-ORGANISM O O O O O O B-GENEPROD O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Expression of p63 in skin epidermis was examined by immunofluorescence staining in WT and cKO mice. (E) Number of p63-positive cells in WT and cKO skin was quantified and shown as box and whisker plots. The plot indicates the mean (solid diamond within the box), 25th percentile (bottom line of the box), median (middle line of the box), 75th percentile (top line of the box), 5th and 95th percentile (whiskers), 1st and 99th percentile (solid triangles) and minimum and maximum measurements (solid squares). n=6 (biological repeats), P<0.05 (Student's t-test).


( F ) Skin stratification in WT and cKO skin was determined by immunofluorescence staining with different antibodies as indicated . Krt14 : Keratin 14 ; Krt10 : Keratin 10 ; β4 : β4 - integrin ; DAPI for nucleus staining . The dashed line denotes the basement membrane that separates dermis and epidermis ( Epi ) .

O O O B-TISSUE O O O O O B-TISSUE O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O B-SUBCELLULAR B-EXP_ASSAY O O O O O O B-TISSUE I-TISSUE O O B-TISSUE O B-TISSUE O B-TISSUE O O

(F) Skin stratification in WT and cKO skin was determined by immunofluorescence staining with different antibodies as indicated. Krt14: Keratin 14; Krt10: Keratin 10; β4: β4-integrin; DAPI for nucleus staining. The dashed line denotes the basement membrane that separates dermis and epidermis (Epi).


( G ) Wound healing as monitored by wound size 8 days post injury . n = 3 ; P < 0 . 01 ( Student ' s t - test ) . Error bar represents S . D . ( standard deviation ) .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-DISEASE O O O O O O O O O O O O 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) Wound healing as monitored by wound size 8 days post injury. n=3; P<0.01 (Student's t-test). Error bar represents S.D. (standard deviation).


H ) Histological staining of skin sections at the wound edges . Halves of wound sections are shown . Note significant reduction of HPE ( hyperproliferative epidermis ) in cKO skin . Es : eschar . Dotted lines denote epidermal boundaries . ( I ) Quantification of Ki67 - positive cells present in wound HPE . The plot indicates the mean ( open circles within the box ) , 25th percentile ( bottom line of the box ) , median ( middle line of the box ) , 75th percentile ( top line of the box ) , 5th and 95th percentile ( whiskers ) , 1st and 99th percentile ( solid triangles ) and minimum and maximum measurements ( solid squares ) . n = 19 , P < 0 . 01 ( Student ' s t - test ) .

O O B-EXP_ASSAY I-EXP_ASSAY O B-TISSUE O O O B-DISEASE O O O O B-DISEASE O O O O O O O O B-TISSUE O B-TISSUE I-TISSUE O O O B-TISSUE O B-TISSUE O B-TISSUE O O O O B-TISSUE O O O O O O O B-GENEPROD O O O O O B-DISEASE B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Histological staining of skin sections at the wound edges. Halves of wound sections are shown. Note significant reduction of HPE (hyperproliferative epidermis) in cKO skin. Es: eschar. Dotted lines denote epidermal boundaries. (I) Quantification of Ki67-positive cells present in wound HPE. The plot indicates the mean (open circles within the box), 25th percentile (bottom line of the box), median (middle line of the box), 75th percentile (top line of the box), 5th and 95th percentile (whiskers), 1st and 99th percentile (solid triangles) and minimum and maximum measurements (solid squares). n=19, P<0.01 (Student's t-test).


( A ) EdU staining of WT or Mettl14 cKO skin after pulse - chase labeling . Skin samples were counterstained with antibody against β4 - integrin . Note reduced EdU label - retaining cells in cKO skin epidermis . Arrows indicate Edu - positive cells . ( B ) Label - retaining cells in WT or Mettl14 cKO skin were quantified and shown as box plots . The plot indicates the mean ( solid diamond within the box ) , 25th percentile ( bottom line of the box ) , median ( middle line of the box ) , 75th percentile ( top line of the box ) , 5th and 95th percentile ( whiskers ) , 1st and 99th percentile ( solid triangles ) and minimum and maximum measurements ( solid squares ) . n = 6 ( biological repeats ) , P < 0 . 05 ( Student ' s t - test ) . n = 19 , P < 0 . 01 ( Student ' s t - test ) .

O O O B-SMALL_MOLECULE B-EXP_ASSAY O O O B-GENEPROD O B-TISSUE O O O O O O B-TISSUE O O B-EXP_ASSAY O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-SMALL_MOLECULE O O O O O O B-TISSUE I-TISSUE O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-GENEPROD O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) EdU staining of WT or Mettl14 cKO skin after pulse-chase labeling. Skin samples were counterstained with antibody against β4-integrin. Note reduced EdU label-retaining cells in cKO skin epidermis. Arrows indicate Edu-positive cells. (B) Label-retaining cells in WT or Mettl14 cKO skin were quantified and shown as box plots. The plot indicates the mean (solid diamond within the box), 25th percentile (bottom line of the box), median (middle line of the box), 75th percentile (top line of the box), 5th and 95th percentile (whiskers), 1st and 99th percentile (solid triangles) and minimum and maximum measurements (solid squares). n=6 (biological repeats), P<0.05 (Student's t-test). n=19, P<0.01 (Student's t-test).


( C ) Number of holoclones derived from WT and Mettl14 cKO skin was quantified and shown as box and whisker plots . The plot indicates the mean ( solid diamond within the box ) , 25th percentile ( bottom line of the box ) , median ( middle line of the box ) , 75th percentile ( top line of the box ) , 5th and 95th percentile ( whiskers ) , 1st and 99th percentile ( solid triangles ) and minimum and maximum measurements ( solid squares ) . n = 6 ( biological repeats ) , P < 0 . 05 ( Student ' s t - test ) . n = 24 , P < 0 . 01 ( Student ' s t - test ) .

O O O O O O O O O O B-GENEPROD O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Number of holoclones derived from WT and Mettl14 cKO skin was quantified and shown as box and whisker plots. The plot indicates the mean (solid diamond within the box), 25th percentile (bottom line of the box), median (middle line of the box), 75th percentile (top line of the box), 5th and 95th percentile (whiskers), 1st and 99th percentile (solid triangles) and minimum and maximum measurements (solid squares). n=6 (biological repeats), P<0.05 (Student's t-test). n=24, P<0.01 (Student's t-test).


( D ) Morphology of primary keratinocytes isolated from WT or Mettl14 cKO skin .

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(D) Morphology of primary keratinocytes isolated from WT or Mettl14 cKO skin.


( E ) Expression of Mettl14 and p63 in WT and KO cells was determined by immunoblots with respective antibodies . Immunoblot for GAPDH was used as loading control .

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(E) Expression of Mettl14 and p63 in WT and KO cells was determined by immunoblots with respective antibodies. Immunoblot for GAPDH was used as loading control.


( F ) Proliferation of WT and Mettl14 null cells in vitro was quantified and shown as dot plots . n = 3 , P < 0 . 01 ( Student ' s t - test ) for Day 6 , 9 , and 12 , and P < 0 . 05 ( Student ' s t - test ) for Day 3 . Error bar represents S . D .

O O O B-EXP_ASSAY O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Proliferation of WT and Mettl14 null cells in vitro was quantified and shown as dot plots. n=3, P<0.01 (Student's t-test) for Day 6, 9, and 12, and P<0.05 (Student's t-test) for Day 3. Error bar represents S.D.


( G - H ) CFE ( colony formation efficiency ) of WT and Mettl14 null cells was determined in vitro . Results were quantified and shown as bar graph ( F ) . n = 19 , P < 0 . 01 ( Student ' s t - test ) . Error bar represents S . D .

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(G-H) CFE (colony formation efficiency) of WT and Mettl14 null cells was determined in vitro. Results were quantified and shown as bar graph (F). n=19, P<0.01 (Student's t-test). Error bar represents S.D.


( I ) Fluorescence microscopy demonstrates different survival capability of WT and Mettl14 inducible KO cells with or without Tamoxifen ( TAM ) treatment . ( J ) Ratio of WT and Mettl14 inducible KO cells in the co - culture model was quantified and shown as dot plots . n = 8 , P < 0 . 01 ( Student ' s t - test ) for KO cells with TAM treatment compared with WT cells or KO cells without TAM stimulation at both Day 7 and 14 . Error bar represents S . D .

O O O B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O 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-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O

(I) Fluorescence microscopy demonstrates different survival capability of WT and Mettl14 inducible KO cells with or without Tamoxifen (TAM) treatment. (J) Ratio of WT and Mettl14 inducible KO cells in the co-culture model was quantified and shown as dot plots. n=8, P<0.01 (Student's t-test) for KO cells with TAM treatment compared with WT cells or KO cells without TAM stimulation at both Day 7 and 14. Error bar represents S.D.


B ) Mutations in the predicted m6A modification sites can significantly reduce Pvt1 methylation . n = 3 . Error bar represents S . D .

O O O O O O B-SMALL_MOLECULE O O O O O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O

B) Mutations in the predicted m6A modification sites can significantly reduce Pvt1 methylation.n=3. Error bar represents S.D.


C ) Deletion of endogenous Pvt1 by an inducible Cas9 ( iCas9 ) system can significantly reduce Pvt1 RNA level ( quantification from RT - PCR , left panel ) and MYC protein level ( quantification from immunoblot , right panel ) expression . n = 3 , P < 0 . 01 ( Student ' s t - test ) . Error bar represents S . D .

O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY O O O O B-EXP_ASSAY O O 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) Deletion of endogenous Pvt1 by an inducible Cas9 (iCas9) system can significantly reduce Pvt1 RNA level (quantification from RT-PCR, left panel) and MYC protein level (quantification from immunoblot, right panel) expression. n=3, P<0.01 (Student's t-test). Error bar represents S.D.


D ) CFE of WT , Pvt1 inducible KO , and Pvt1 inducible KO cells rescued with WT or mutant Pvt1 was quantified and presented as bar graph . n = 3 , * * : P < 0 . 01 ( Student ' s t - test ) . Error bar represents S . D .

O O B-EXP_ASSAY O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O O O O O 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) CFE of WT, Pvt1 inducible KO, and Pvt1 inducible KO cells rescued with WT or mutant Pvt1 was quantified and presented as bar graph. n=3, **: P<0.01 (Student's t-test). Error bar represents S.D.


E ) Skin organoids derived from WT or Pvt1 inducible KO cells were grafted to nude mice . The regenerated skin was analyzed by H / E staining .

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E) Skin organoids derived from WT or Pvt1 inducible KO cells were grafted to nude mice. The regenerated skin was analyzed by H/E staining.


F ) Epidermal thickness of WT and Pvt1 KO skin grafts was quantified and presented as box and whisker plots . The plot indicates the mean ( solid diamond within the box ) , 25th percentile ( bottom line of the box ) , median ( middle line of the box ) , 75th percentile ( top line of the box ) , 5th and 95th percentile ( whiskers ) , 1st and 99th percentile ( solid triangles ) and minimum and maximum measurements ( solid squares ) . n = 6 ( biological repeats ) , P < 0 . 05 ( Student ' s t - test ) . n = 18 , P < 0 . 01 ( Student ' s t - test ) .

O O B-TISSUE O O O O B-GENEPROD O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Epidermal thickness of WT and Pvt1 KO skin grafts was quantified and presented as box and whisker plots. The plot indicates the mean (solid diamond within the box), 25th percentile (bottom line of the box), median (middle line of the box), 75th percentile (top line of the box), 5th and 95th percentile (whiskers), 1st and 99th percentile (solid triangles) and minimum and maximum measurements (solid squares). n=6 (biological repeats), P<0.05 (Student's t-test). n=18, P<0.01 (Student's t-test).


G ) Number of p63 - positive cells in WT and cKO skin was quantified and shown as bar graph . n = 5 , P < 0 . 01 ( Student ' s t - test ) . Error bar represents S . D .

O O O O B-GENEPROD O O O O O O O B-TISSUE O O O O O O O O O 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) Number of p63-positive cells in WT and cKO skin was quantified and shown as bar graph. n=5, P<0.01 (Student's t-test). Error bar represents S.D.


H ) Fluorescence microscopy demonstrates different survival capability of WT and Pvt1 inducible KO cells with or without Doxycycline ( Dox ) treatment .

O O B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O

H) Fluorescence microscopy demonstrates different survival capability of WT and Pvt1 inducible KO cells with or without Doxycycline (Dox) treatment.


I ) Ratio of WT and Pvt1 inducible KO cells in the co - culture model was quantified and shown as dot plots . n = 8 , P < 0 . 01 ( Student ' s t - test ) for KO cells with Dox treatment compared with WT cells or KO cells without Dox stimulation at both Day 7 and 14 . Error bar represents S . D .

O O O O O O B-GENEPROD O O O O O O O O O O O O O O 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-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O

I) Ratio of WT and Pvt1 inducible KO cells in the co-culture model was quantified and shown as dot plots. n=8, P<0.01 (Student's t-test) for KO cells with Dox treatment compared with WT cells or KO cells without Dox stimulation at both Day 7 and 14. Error bar represents S.D.


A ) Interaction between Pvt1 and MYC was determined by immunoprecipitation followed with RT - PCR . Note significant decrease in Pvt1 and MYC interaction upon FTO treatment ( left panel ) , calcium shift - induced differentiation ( middle panel , Hi : high calcium ) , and Pvt1 methylation site mutations ( right panel ) . n = 3 , P < 0 . 01 ( Student ' s t - test ) . Error bar represents S . D .

O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O O O B-GENEPROD B-EXP_ASSAY O O O O O O O O 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) Interaction between Pvt1 and MYC was determined by immunoprecipitation followed with RT-PCR. Note significant decrease in Pvt1 and MYC interaction upon FTO treatment (left panel), calcium shift-induced differentiation (middle panel, Hi: high calcium), and Pvt1 methylation site mutations (right panel). n=3, P<0.01 (Student's t-test). Error bar represents S.D.


B ) Immunoblots show decreased MYC protein level in differentiated keratinocytes and Mettl14 KO cells .

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B) Immunoblots show decreased MYC protein level in differentiated keratinocytes and Mettl14 KO cells.


Undifferentiated WT and Mettl14 KO cells ( C ) were treated with cycloheximide . Cell lysates were collected at 0 , 30 , 60 , 90 , 120 , 180 , and 300 minutes post treatment and subjected to immunoblotting with different antibodies as indicated .

O O O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE O O 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-EXP_ASSAY O O O O O O

Undifferentiated WT and Mettl14 KO cells (C) were treated with cycloheximide. Cell lysates were collected at 0, 30, 60, 90, 120, 180, and 300 minutes post treatment and subjected to immunoblotting with different antibodies as indicated.


undifferentiated WT and Pvt1 KO cells ( D ) were treated with cycloheximide . Cell lysates were collected at 0 , 30 , 60 , 90 , 120 , 180 , and 300 minutes post treatment and subjected to immunoblotting with different antibodies as indicated .

undifferentiated WT and Pvt1 KO cells (D) were treated with cycloheximide. Cell lysates were collected at 0, 30, 60, 90, 120, 180, and 300 minutes post treatment and subjected to immunoblotting with different antibodies as indicated.


WT cells before and after calcium shift - induced differentiation ( E ) were treated with cycloheximide . Cell lysates were collected at 0 , 30 , 60 , 90 , 120 , 180 , and 300 minutes post treatment and subjected to immunoblotting with different antibodies as indicated .

O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O 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-EXP_ASSAY O O O O O O

WT cells before and after calcium shift-induced differentiation (E) were treated with cycloheximide. Cell lysates were collected at 0, 30, 60, 90, 120, 180, and 300 minutes post treatment and subjected to immunoblotting with different antibodies as indicated.


( F ) Skin organoids derived from Mettl14 KO cells or KO cells rescued with exogenous expression of MYC were grafted to nude mice . The regenerated skin was analyzed by H / E staining .

O O O B-TISSUE O O O B-GENEPROD O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O B-ORGANISM O O O B-TISSUE O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O

(F) Skin organoids derived from Mettl14 KO cells or KO cells rescued with exogenous expression of MYC were grafted to nude mice. The regenerated skin was analyzed by H/E staining.


( G ) Epidermal thickness of KO and KO skin rescued with MYC expression was quantified and presented as box and whisker plots . The plot indicates the mean ( solid diamond within the box ) , 25th percentile ( bottom line of the box ) , median ( middle line of the box ) , 75th percentile ( top line of the box ) , 5th and 95th percentile ( whiskers ) , 1st and 99th percentile ( solid triangles ) and minimum and maximum measurements ( solid squares ) . n = 6 ( biological repeats ) , P < 0 . 05 ( Student ' s t - test ) . n = 18 , P < 0 . 01 ( Student ' s t - test ) .

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(G) Epidermal thickness of KO and KO skin rescued with MYC expression was quantified and presented as box and whisker plots. The plot indicates the mean (solid diamond within the box), 25th percentile (bottom line of the box), median (middle line of the box), 75th percentile (top line of the box), 5th and 95th percentile (whiskers), 1st and 99th percentile (solid triangles) and minimum and maximum measurements (solid squares). n=6 (biological repeats), P<0.05 (Student's t-test). n=18, P<0.01 (Student's t-test).


( H ) Number of p63 - positive cells in KO and KO skin rescued with MYC expression was quantified and shown as bar graph . n = 5 , P < 0 . 01 ( Student ' s t - test ) . Error bar represents S . D .

O O O O O B-GENEPROD O O O O O O O B-TISSUE O O B-GENEPROD O O O O O O O O O O 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) Number of p63-positive cells in KO and KO skin rescued with MYC expression was quantified and shown as bar graph. n=5, P<0.01 (Student's t-test). Error bar represents S.D.


( I ) Fluorescence microscopy demonstrates different survival capability of WT and Mettl14 KO cells with inducible expression of MYC .

O O O B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O O O O B-GENEPROD O

(I) Fluorescence microscopy demonstrates different survival capability of WT and Mettl14 KO cells with inducible expression of MYC.


( J ) Ratio of WT and Mettl14 KO cells with inducible expression of MYC in the co - culture model was quantified and shown as dot plots . n = 8 , P < 0 . 01 ( Student ' s t - test ) for KO cells without Dox treatment ( no MYC expression ) compared with WT cells or KO cells with Dox stimulation ( exogenous MYC expression ) at both Day 7 and 14 . Error bar represents S . D .

O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O 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-SMALL_MOLECULE O O O B-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE O O O B-GENEPROD O O O O O O O O O O O O O O O O

(J) Ratio of WT and Mettl14 KO cells with inducible expression of MYC in the co-culture model was quantified and shown as dot plots. n=8, P<0.01 (Student's t-test) for KO cells without Dox treatment (no MYC expression) compared with WT cells or KO cells with Dox stimulation (exogenous MYC expression) at both Day 7 and 14. Error bar represents S.D.


( A ) Western blot analysis showing the expression of the WRNIP1 protein in wild - type cells ( shWRNIP1WT ) and WRNIP1 - deficient ( shWRNIP1 ) or mutant ( shWRNIP1T294A ) cells . MRC5SV fibroblasts were used as a positive control . The membrane was probed with an anti - FLAG or anti - WRNIP1 . GAPDH was used as a loading control . Below each lane of the blot the ratio of WRNIP1 protein to total protein , then normalised to MRC5SV , is reported .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O B-CELL_LINE O O O O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O B-CELL_LINE O O O O

(A) Western blot analysis showing the expression of the WRNIP1 protein in wild-type cells (shWRNIP1WT) and WRNIP1-deficient (shWRNIP1) or mutant (shWRNIP1T294A) cells. MRC5SV fibroblasts were used as a positive control. The membrane was probed with an anti-FLAG or anti-WRNIP1. GAPDH was used as a loading control. Below each lane of the blot the ratio of WRNIP1 protein to total protein, then normalised to MRC5SV, is reported.


( B ) Experimental scheme of dual labelling of DNA fibers in shWRNIP1WT , shWRNIP1 and shWRNIP1T294A cells . Cells were pulse - labelled with CldU , and then subjected to a pulse - labelling with IdU . ( C ) Analysis of replication fork velocity ( fork speed ) in the cells under unperturbed conditions . The length of the green tracks were measured . Mean values are represented as horizontal black lines ( ns , not significant ; Student ' s t - test ) .

O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O O 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) Experimental scheme of dual labelling of DNA fibers in shWRNIP1WT, shWRNIP1 and shWRNIP1T294A cells. Cells were pulse-labelled with CldU, and then subjected to a pulse-labelling with IdU.(C) Analysis of replication fork velocity (fork speed) in the cells under unperturbed conditions. The length of the green tracks were measured. Mean values are represented as horizontal black lines (ns, not significant; Student's t-test).


( D ) Cells were treated as in ( B ) . For each replication origin , the length of the right - fork signal was measured and plotted against the length of the left - fork signal . A schematic representation of symmetric and asymmetric forks is given . If the ratio between the left - fork length and the right - fork length deviated by more than 33 % from 1 ( that is , outside the violet dashed lines in the graphs ) , the fork was considered asymmetric . The percentage of asymmetric forks was calculated for all cell lines . N = number of forks counted for each cell line . R represents linear correlation coefficient .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Cells were treated as in (B). For each replication origin, the length of the right-fork signal was measured and plotted against the length of the left-fork signal. A schematic representation of symmetric and asymmetric forks is given. If the ratio between the left-fork length and the right-fork length deviated by more than 33% from 1 (that is, outside the violet dashed lines in the graphs), the fork was considered asymmetric. The percentage of asymmetric forks was calculated for all cell lines. N= number of forks counted for each cell line. R represents linear correlation coefficient.


( E ) Experimental scheme of dual labelling of DNA fibers in shWRNIP1WT , shWRNIP1 and shWRNIP1T294A cells . Cells were pulse - labelled with CldU , treated with 4mM HU and then subjected to a pulse - labelling with IdU . ( F ) Graphs show the percentage of red ( CldU ) tracts ( stalled forks ) or red - green ( CldU - IdU ) contiguous tracts ( restarting forks ) in the cells . Mean shown , n = 3 . Error bars represent standard error . ( * , p < 0 . 05 ; * * , p < 0 . 01 ; Student ' s t test ) . Representative DNA fiber images are shown . Scale bars , 10 µm .

O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O O O O O O O O O O O

(E) Experimental scheme of dual labelling of DNA fibers in shWRNIP1WT, shWRNIP1 and shWRNIP1T294A cells. Cells were pulse-labelled with CldU, treated with 4mM HU and then subjected to a pulse-labelling with IdU.(F) Graphs show the percentage of red (CldU) tracts (stalled forks) or red-green (CldU-IdU) contiguous tracts (restarting forks) in the cells. Mean shown, n = 3. Error bars represent standard error. (*, p < 0.05; **, p < 0.01; Student's t test). Representative DNA fiber images are shown. Scale bars, 10 µm.


( G ) Experimental scheme of dual labelling of DNA fibers in shWRNIP1WT , shWRNIP1 and shWRNIP1T294A cells . Cells were sequentially pulse - labelled with CldU and IdU as indicated , then treated or not with 4 mM HU . ( H ) Representative IdU tract length distributions in all cell lines under unperturbed conditions ( left graph ) or after HU treatment ( right graph ) . Median tract lengths are given in parentheses . See also Tables S1 and S2 for details on the data sets and statistical test . Representative DNA fiber images are shown . Scale bars , 10 µm .

O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O 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-SMALL_MOLECULE O O O O O O O O O O O

(G) Experimental scheme of dual labelling of DNA fibers in shWRNIP1WT, shWRNIP1 and shWRNIP1T294A cells. Cells were sequentially pulse-labelled with CldU and IdU as indicated, then treated or not with 4 mM HU.(H) Representative IdU tract length distributions in all cell lines under unperturbed conditions (left graph) or after HU treatment (right graph). Median tract lengths are given in parentheses. See also Tables S1 and S2 for details on the data sets and statistical test. Representative DNA fiber images are shown. Scale bars, 10 µm.


( A ) Experimental scheme of dual labelling of DNA fibers in wild - type cells ( shWRNIP1WT ) or WRNIP1 - deficient cells ( shWRNIP1 ) . Cells were sequentially pulse - labelled with CldU and IdU as indicated , then left untreated or treated with 4 mM HU in combination or not with 50 µM Mirin . ( B ) Representative IdU tract length distributions in shWRNIP1WT ( left graph ) or shWRNIP1 cells ( right graph ) after treatment . Median tract lengths are given in parentheses . See Tables S1 and S2 for details on the data sets and statistical test . Representative DNA fiber images are shown . Scale bars , 10 µm .

O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O O O O O O O O O O O

(A) Experimental scheme of dual labelling of DNA fibers in wild-type cells (shWRNIP1WT) or WRNIP1-deficient cells (shWRNIP1). Cells were sequentially pulse-labelled with CldU and IdU as indicated, then left untreated or treated with 4 mM HU in combination or not with 50 µM Mirin.(B) Representative IdU tract length distributions in shWRNIP1WT (left graph) or shWRNIP1 cells (right graph) after treatment. Median tract lengths are given in parentheses. See Tables S1 and S2 for details on the data sets and statistical test. Representative DNA fiber images are shown. Scale bars, 10 µm.


( A ) Evaluation of ssDNA accumulation at parental - strand by immunofluorescence microscopy analysis in wild - type ( shWRNIP1WT ) or WRNIP1 - deficient ( shWRNIP1 ) cells . Experimental design of ssDNA assay is shown . Cells were labelled with IdU for 24 h , as indicated , washed and left to recover for 2 h , then treated or not with 4 mM HU . In parallel samples , the MRE11 activity is chemically inhibited with 50 µM Mirin , alone or in combination with HU - induced replication stress . After treatment , cells were fixed and stained with an anti - IdU antibody without denaturing the DNA to specifically detect parental ssDNA . Horizontal black lines represent the mean SE ; n = 3 . Error bars represent standard error ( ns , not significant ; * * , p < 0 . 01 ; * * * * , p < 0 . 0001 ; two - tailed Student ' s t test ) . Representative images are shown . DNA was counterstained with DAPI ( blue ) .

O O O O O B-SMALL_MOLECULE O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O O O O O O O O

(A) Evaluation of ssDNA accumulation at parental-strand by immunofluorescence microscopy analysis in wild-type (shWRNIP1WT) or WRNIP1-deficient (shWRNIP1) cells. Experimental design of ssDNA assay is shown. Cells were labelled with IdU for 24 h, as indicated, washed and left to recover for 2 h, then treated or not with 4 mM HU. In parallel samples, the MRE11 activity is chemically inhibited with 50 µM Mirin, alone or in combination with HU-induced replication stress. After treatment, cells were fixed and stained with an anti-IdU antibody without denaturing the DNA to specifically detect parental ssDNA. Horizontal black lines represent the mean SE; n = 3. Error bars represent standard error (ns, not significant; **, p<0.01; ****, p<0.0001; two-tailed Student's t test). Representative images are shown. DNA was counterstained with DAPI (blue).


( B ) Analysis of chromatin binding of MRE11 and RAD51 in shWRNIP1WT and shWRNIP1 cells . Chromatin fractions of cells , treated or not with 4 mM HU , were analysed by immunoblotting . The membrane was probed with the anti - WRNIP1 , anti - MRE11 and anti - RAD51 antibodies . LAMIN B1 was used as a loading for the chromatin fraction . Total amount of RAD51 and MRE11 ( Input ) in the cells was determined with the relevant antibodies . LAMIN B1 was used as a loading control . In the graph , the fold increase respect to the wild - type untreated of the normalized ratio of the chromatin - bound RAD51 ( or MRE11 ) / total RAD51 ( or MRE11 ) is reported for each cell line .

O O O O O B-SUBCELLULAR O O B-GENEPROD O B-GENEPROD O O O O O O B-SUBCELLULAR O O O O O O O O O O B-SMALL_MOLECULE O O O O B-EXP_ASSAY O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O B-GENEPROD I-GENEPROD O O O O O O O B-SUBCELLULAR O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD I-GENEPROD O O O 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-SUBCELLULAR O O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD O O B-GENEPROD O O O O O O O O

(B) Analysis of chromatin binding of MRE11 and RAD51 in shWRNIP1WT and shWRNIP1 cells. Chromatin fractions of cells, treated or not with 4 mM HU, were analysed by immunoblotting. The membrane was probed with the anti-WRNIP1, anti-MRE11 and anti-RAD51 antibodies. LAMIN B1 was used as a loading for the chromatin fraction. Total amount of RAD51 and MRE11 (Input) in the cells was determined with the relevant antibodies. LAMIN B1 was used as a loading control. In the graph, the fold increase respect to the wild-type untreated of the normalized ratio of the chromatin-bound RAD51 (or MRE11)/ total RAD51 (or MRE11) is reported for each cell line.


( C ) Analysis of DNA - protein interactions between ssDNA and endogenous RAD51 in shWRNIP1WT and shWRNIP1 cells by in situ PLA assay . Experimental designed used for the assay is given . Cells were labelled with IdU for 24 h , as indicated , washed and left to recover for 2 h , then treated or not with 4 mM HU for 4 h . Next , cells were fixed , stained with an anti - IdU antibody without denaturing the DNA to specifically detect parental - strand ssDNA , and subjected to PLA assay as described in the " Experimental procedures " section . Antibodies raised against IdU or RAD51 were used to reveal ssDNA or endogenous RAD51 respectively . Each red spot represents a single interaction between ssDNA and RAD51 . No spot has been revealed in cells stained with each single antibody ( negative control ) . DNA was counterstained with DAPI ( blue ) . Representative images of the PLA assay are given . Graph shows the number of PLA spots per cell . Horizontal black lines represent the mean value ( ns , not significant ; * * , p < 0 . 01 ; two - tailed Student ' s t test ) ; n = 3 .

O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O B-GENEPROD O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O B-EXP_ASSAY O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-GENEPROD O O O O B-SMALL_MOLECULE O O B-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE O B-GENEPROD O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O B-EXP_ASSAY O O O O O O O O B-EXP_ASSAY O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O 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) Analysis of DNA-protein interactions between ssDNA and endogenous RAD51 in shWRNIP1WT and shWRNIP1 cells by in situ PLA assay. Experimental designed used for the assay is given. Cells were labelled with IdU for 24 h, as indicated, washed and left to recover for 2 h, then treated or not with 4 mM HU for 4 h. Next, cells were fixed, stained with an anti-IdU antibody without denaturing the DNA to specifically detect parental-strand ssDNA, and subjected to PLA assay as described in the "Experimental procedures" section. Antibodies raised against IdU or RAD51 were used to reveal ssDNA or endogenous RAD51 respectively. Each red spot represents a single interaction between ssDNA and RAD51. No spot has been revealed in cells stained with each single antibody (negative control). DNA was counterstained with DAPI (blue). Representative images of the PLA assay are given. Graph shows the number of PLA spots per cell. Horizontal black lines represent the mean value (ns, not significant; **, p < 0.01; two-tailed Student's t test); n = 3.


( D ) Localization of WRNIP1 , MRE11 and RAD51 to stalled replication forks . Forks were isolated by CldU - co - immunoprecipitation ( CldU - IP ) . shWRNIP1WT or shWRNIP1 cells were pulse - labelled with CldU , then fixed or treated with HU . Cells were cross - linked , and the nuclear extracts were isolated ( Input ) and subjected to CldU - IP using an anti - CldU antibody ( CldU - IP ) . The membranes were probed with the anti - WRNIP1 or anti - RAD51 antibodies . After stripping , the membranes were probed with an anti - MRE11 antibody . LAMIN B1 and GAPDH were used as loading controls ( Input ) . Ponceau S was used as a loading control of CldU - IP . Dot blot analysis was performed to confirm that equal amounts of immunoprecipitatedDNA from each sample . 10 % of each IP was loaded on a nitrocellulose membrane . The membrane was probed with an anti - CldU antibody . The graph shows the normalized ratio of the proteins co - immunoprecipitated with CldU ( CldUCo - IP proteins ) / the total of labelled DNAimmunoprecipitated with CldU ( CldUIP ) for each cell line after replication stress from two independent experiments . The dots in the graph represent the individual data points from each single experiment . Horizontal black line represents the mean value from two replicates ; n = 2 .

O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-SUBCELLULAR I-SUBCELLULAR O O O O O B-SMALL_MOLECULE O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O B-EXP_ASSAY O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-SUBCELLULAR O O O O O O O O O B-SMALL_MOLECULE O B-EXP_ASSAY O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-EXP_ASSAY O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-SMALL_MOLECULE O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-EXP_ASSAY O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O B-SMALL_MOLECULE B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O 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) Localization of WRNIP1, MRE11 and RAD51 to stalled replication forks. Forks were isolated by CldU-co-immunoprecipitation (CldU-IP). shWRNIP1WT or shWRNIP1 cells were pulse-labelled with CldU, then fixed or treated with HU. Cells were cross-linked, and the nuclear extracts were isolated (Input) and subjected to CldU-IP using an anti-CldU antibody (CldU-IP). The membranes were probed with the anti-WRNIP1 or anti-RAD51 antibodies. After stripping, the membranes were probed with an anti-MRE11 antibody. LAMIN B1 and GAPDH were used as loading controls (Input). Ponceau S was used as a loading control of CldU-IP. Dot blot analysis was performed to confirm that equal amounts of immunoprecipitatedDNA from each sample. 10% of each IP was loaded on a nitrocellulose membrane. The membrane was probed with an anti-CldU antibody. The graph shows the normalized ratio of the proteins co-immunoprecipitated with CldU (CldUCo-IP proteins)/ the total of labelled DNAimmunoprecipitated with CldU (CldUIP) for each cell line after replication stress from two independent experiments. The dots in the graph represent the individual data points from each single experiment. Horizontal black line represents the mean value from two replicates; n = 2.


( A ) Experimental scheme of pulse - labelling of DNA fibers in wild - type cells ( shWRNIP1WT ) or WRNIP1 - deficient cells ( shWRNIP1 ) . Cells were labelled with IdU and exposed or not to 25 μM RAD51 inhibitor , then treated or not with 4 mM HU . ( B ) Representative IdU tract length distributions in shWRNIP1WT cells ( left graph ) or shWRNIP1 cells ( right graph ) . Median tract lengths are reported in parentheses . See Tables S1 and S2 for details on the data sets and statistical test . Representative DNA fiber images are reported . Scale bars , 10 µm .

O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O O O O O O O O O O O

(A) Experimental scheme of pulse-labelling of DNA fibers in wild-type cells (shWRNIP1WT) or WRNIP1-deficient cells (shWRNIP1). Cells were labelled with IdU and exposed or not to 25 μM RAD51 inhibitor, then treated or not with 4 mM HU.(B) Representative IdU tract length distributions in shWRNIP1WT cells (left graph) or shWRNIP1 cells (right graph). Median tract lengths are reported in parentheses. See Tables S1 and S2 for details on the data sets and statistical test. Representative DNA fiber images are reported. Scale bars, 10 µm.


( C ) Scheme of DNA fiber tract analysis in shWRNIP1 cells . Cells were transfected with an empty vector or a plasmid expressing a wild - type human RAD51 , and 48 h thereafter labelled with IdU and treated or not with 4 mM HU . ( D ) Representative IdU tract length distributions in shWRNIP1 cells or shWRNIP1 cells expressing exogenous wild - type RAD51 after HU exposure . Median tract lengths are given in parentheses . See Tables S1 and S2 for details on the data sets and statistical test . Representative DNA fiber images are given . Scale bars , 10 µm . Western blot shows the expression of the RAD51 protein in shWRNIP1 cells . The membrane was probed with an anti - RAD51 . LAMIN B1 was used as a loading control .

O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE O O 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-SMALL_MOLECULE O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O O O O O O O

(C) Scheme of DNA fiber tract analysis in shWRNIP1 cells. Cells were transfected with an empty vector or a plasmid expressing a wild-type human RAD51, and 48 h thereafter labelled with IdU and treated or not with 4 mM HU.(D) Representative IdU tract length distributions in shWRNIP1 cells or shWRNIP1 cells expressing exogenous wild-type RAD51 after HU exposure. Median tract lengths are given in parentheses. See Tables S1 and S2 for details on the data sets and statistical test. Representative DNA fiber images are given. Scale bars, 10 µm. Western blot shows the expression of the RAD51 protein in shWRNIP1 cells. The membrane was probed with an anti-RAD51. LAMIN B1 was used as a loading control.


( A ) Co - immunoprecipitation experiments in HEK293T cells transfected with empty vector or FLAG - WRNIP1 plasmid . Cells were treated or not with HU . After treatment , cell lysates were immunoprecipitated ( FLAG - IP ) using anti - FLAG antibody . The presence of WRNIP1 , BRCA2 and RAD51 was assessed by immunoblotting using the anti - FLAG , anti - RAD51 and anti - BRCA2 antibodies , respectively . Whole cell extracts were analysed ( Input ) . The membrane was probed with the same antibodies used for IP . GAPDH was used as a loading control .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-CELL_LINE O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-EXP_ASSAY O O O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY O O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O

(A) Co-immunoprecipitation experiments in HEK293T cells transfected with empty vector or FLAG-WRNIP1 plasmid. Cells were treated or not with HU. After treatment, cell lysates were immunoprecipitated (FLAG-IP) using anti-FLAG antibody. The presence of WRNIP1, BRCA2 and RAD51 was assessed by immunoblotting using the anti-FLAG, anti-RAD51 and anti-BRCA2 antibodies, respectively. Whole cell extracts were analysed (Input). The membrane was probed with the same antibodies used for IP. GAPDH was used as a loading control.


( B ) Analysis of protein - protein interactions between WRNIP1 and endogenous RAD51 in wild - type ( shWRNIP1WT ) or WRNIP1 - mutant ( shWRNIP1T294A ) cells by in situ PLA assay . Cells were labelled with IdU for 24 h , washed and left to recover for 2 h , then treated or not with 4 mM HU . Antibodies raised against FLAG - Tag and RAD51 were used to reveal FLAG - WRNIP1 or endogenous RAD51 respectively . Each red spot represents a single interaction between WRNIP1 and RAD51 . No spot has been revealed in cells stained with each single antibody ( negative control ) . DNA was counterstained with DAPI ( blue ) . Representative images of the PLA assay are shown . Graph shows the mean number of PLA spots per cell SE . Error bars represent standard error ( ns , not significant ; two - tailed Student ' s t test ) ; n = 3 .

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(B) Analysis of protein-protein interactions between WRNIP1 and endogenous RAD51 in wild-type (shWRNIP1WT) or WRNIP1-mutant (shWRNIP1T294A) cells by in situ PLA assay. Cells were labelled with IdU for 24 h, washed and left to recover for 2 h, then treated or not with 4 mM HU. Antibodies raised against FLAG-Tag and RAD51 were used to reveal FLAG-WRNIP1 or endogenous RAD51 respectively. Each red spot represents a single interaction between WRNIP1 and RAD51. No spot has been revealed in cells stained with each single antibody (negative control). DNA was counterstained with DAPI (blue). Representative images of the PLA assay are shown. Graph shows the mean number of PLA spots per cell SE. Error bars represent standard error (ns, not significant; two-tailed Student's t test); n = 3.


( C ) Experimental scheme of pulse - labelling of DNA fibers in wild - type cells ( shWRNIP1WT ) or WRNIP1 - deficient cells ( shWRNIP1 ) . Cells were transfected with BRCA2 siRNA ( siBRCA2 ) , and 48 h thereafter labelled with IdU , then treated or not with 4 mM HU . ( D ) Representative IdU tract length distributions in shWRNIP1WT / siBRCA2 or shWRNIP1siBRCA2 cells treated or not with HU . Median tract lengths are given in parentheses . See Tables S1 and S2 for details on the data sets and statistical test . Representative DNA fiber images are reported . Scale bars , 10 µm . Western blot shows BRCA2 depletion in shWRNIP1WT and shWRNIP1 cells . The membrane was probed with an anti - BRCA2 or anti - WRNIP1 . GAPDH was used as a loading control .

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(C) Experimental scheme of pulse-labelling of DNA fibers in wild-type cells (shWRNIP1WT) or WRNIP1-deficient cells (shWRNIP1). Cells were transfected with BRCA2 siRNA (siBRCA2), and 48 h thereafter labelled with IdU, then treated or not with 4 mM HU.(D) Representative IdU tract length distributions in shWRNIP1WT/siBRCA2 or shWRNIP1siBRCA2 cells treated or not with HU. Median tract lengths are given in parentheses. See Tables S1 and S2 for details on the data sets and statistical test. Representative DNA fiber images are reported. Scale bars, 10 µm. Western blot shows BRCA2 depletion in shWRNIP1WT and shWRNIP1 cells. The membrane was probed with an anti-BRCA2 or anti-WRNIP1. GAPDH was used as a loading control.


( E ) Experimental scheme of pulse - labelling of DNA fibers in shWRNIP1 cells . Cells were transfected with control siRNA ( shWRNIP1siCtrl ) or FBH1 siRNA ( shWRNIP1siFBH1 ) , and 48 h thereafter labelled with IdU , then treated or not with 4 mM HU . ( F ) Representative IdU tract length distributions in shWRNIP1siCtrl or shWRNIP1siFBH1 cells with or without HU treatment . Representative DNA fiber images are reported . Scale bars , 10 µm . Western blot shows FBH1 depletion in the cells . The membrane was probed with an anti - FBH1 . GAPDH was used as a loading control . Median tract lengths are given in parentheses . See Tables S1 and S2 for details on the data sets and statistical test .

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(E) Experimental scheme of pulse-labelling of DNA fibers in shWRNIP1 cells. Cells were transfected with control siRNA (shWRNIP1siCtrl) or FBH1 siRNA (shWRNIP1siFBH1), and 48 h thereafter labelled with IdU, then treated or not with 4 mM HU.(F) Representative IdU tract length distributions in shWRNIP1siCtrl or shWRNIP1siFBH1 cells with or without HU treatment. Representative DNA fiber images are reported. Scale bars, 10 µm. Western blot shows FBH1 depletion in the cells. The membrane was probed with an anti-FBH1. GAPDH was used as a loading control. Median tract lengths are given in parentheses. See Tables S1 and S2 for details on the data sets and statistical test.


( G ) Analysis of chromatin binding of RAD51 in shWRNIP1 cells depleted for FBH1 . Cells were transfected with control siRNA ( shWRNIP1siCtrl ) or FBH1 siRNA ( shWRNIP1siFBH1 ) , and 48 h treated or not with HU for 4h . Chromatin fractions of cells were analysed by immunoblotting . The membrane was probed with the anti - FBH1 and anti - RAD51 antibodies . LAMIN B1 was used as a loading for the chromatin fraction . Total amount of RAD51 ( Input ) in the cells was determined with the relevant antibodies . GAPDH was used as a loading control . The ratio of the RAD51 / LAMIN B1 signal ( chromatin ) is reported below each lane .

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(G) Analysis of chromatin binding of RAD51 in shWRNIP1 cells depleted for FBH1. Cells were transfected with control siRNA (shWRNIP1siCtrl) or FBH1 siRNA (shWRNIP1siFBH1), and 48 h treated or not with HU for 4h. Chromatin fractions of cells were analysed by immunoblotting. The membrane was probed with the anti-FBH1 and anti-RAD51 antibodies. LAMIN B1 was used as a loading for the chromatin fraction. Total amount of RAD51 (Input) in the cells was determined with the relevant antibodies. GAPDH was used as a loading control. The ratio of the RAD51/LAMIN B1 signal (chromatin) is reported below each lane.


( A ) Analysis of DNA damage accumulation . Wild - type ( shWRNIP1WT ) , WRNIP1 - deficient ( shWRNIP1 ) or mutant ( shWRNIP1T294A ) cells were treated or not with 4 mM HU for 4 h , then subjected to - H2AXimmunofluorescence microscopy . Graph shows data presented as mean of - H2AX - positive cells SE from three independent experiments ; n = 3 . Error bars represent standard error ( * , p < 0 . 1 ; * * , p < 0 . 01 ; two - tailed Student ' s t test ) . Representative images of nuclei showing the different number of foci per nucleus are reported .

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(A) Analysis of DNA damage accumulation. Wild-type (shWRNIP1WT), WRNIP1-deficient (shWRNIP1) or mutant (shWRNIP1T294A) cells were treated or not with 4 mM HU for 4 h, then subjected to -H2AXimmunofluorescence microscopy. Graph shows data presented as mean of -H2AX-positive cells SE from three independent experiments; n = 3. Error bars represent standard error (*, p < 0.1; **, p < 0.01; two-tailed Student's t test). Representative images of nuclei showing the different number of foci per nucleus are reported.


( B ) Analysis of DNA breakage accumulation . shWRNIP1WT , shWRNIP1 and shWRNIP1T294A cells were treated as in ( A ) , then subjected to alkaline Comet assay . Graph shows data presented as mean tail moment SE from three independent experiments ; n = 3 . Error bars represent standard error ( * , p < 0 . 1 ; * * , p < 0 . 01 ; two - tailed Student ' s t test ) . Representative images are shown .

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(B) Analysis of DNA breakage accumulation. shWRNIP1WT, shWRNIP1 and shWRNIP1T294A cells were treated as in (A), then subjected to alkaline Comet assay. Graph shows data presented as mean tail moment SE from three independent experiments; n = 3. Error bars represent standard error (*, p < 0.1; **, p < 0.01; two-tailed Student's t test). Representative images are shown.


( C ) Evaluation of cell death . shWRNIP1WT shWRNIP1 and shWRNIP1T294A cells were treated or not with 4 mM HU for 16 h . Cell viability was evaluated by LIVE / DEAD fluorescent assay . Data are expressed as mean of dead cells SE from three independent experiments ; n = 3 . Error bars represent standard error ( * , p < 0 . 1 ; * * , p < 0 . 01 ; two - tailed Student ' s t test ) . Representative images of double - staining of viable ( green ) and dead ( red ) cells are shown .

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(C) Evaluation of cell death. shWRNIP1WT shWRNIP1 and shWRNIP1T294A cells were treated or not with 4 mM HU for 16 h. Cell viability was evaluated by LIVE/DEAD fluorescent assay. Data are expressed as mean of dead cells SE from three independent experiments; n = 3. Error bars represent standard error (*, p < 0.1; **, p < 0.01; two-tailed Student's t test). Representative images of double-staining of viable (green) and dead (red) cells are shown.


( D ) Experimental scheme for evaluation of the chromosomal aberrations is shown . shWRNIP1WT shWRNIP1 and shWRNIP1T294A cells were treated or not with 4 mM HU , then left to recover for 16h in drug - free medium and metaphases collected with colcemid . Next , cells were fixed and processed as reported in " Supplemental information " section . Dot plot shows the number of chromosomal aberrations per cell . Horizontal black lines represent the mean SE . Error bars represent standard error ( ns , not significant ; * * , p < 0 . 01 ; two - tailed Student ' s t test ) . Representative Giemsa - stained metaphases of cells treated or not with 4 mM HU . Arrows indicate chromosomal aberrations .

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(D) Experimental scheme for evaluation of the chromosomal aberrations is shown. shWRNIP1WT shWRNIP1 and shWRNIP1T294A cells were treated or not with 4 mM HU, then left to recover for 16h in drug-free medium and metaphases collected with colcemid. Next, cells were fixed and processed as reported in "Supplemental information" section. Dot plot shows the number of chromosomal aberrations per cell. Horizontal black lines represent the mean SE. Error bars represent standard error (ns, not significant; **, p < 0.01; two-tailed Student's t test). Representative Giemsa-stained metaphases of cells treated or not with 4 mM HU. Arrows indicate chromosomal aberrations.


( E ) Experimental scheme of the Mirin aberration analysis is given . The experiment was carried out as in ( D ) but cells were pre - treated or not with 50 µM Mirin . Dot plot shows the effect of Mirin exposure on the number of chromosome aberrations per cell in shWRNIP1 cells . Horizontal black lines represent the mean SE . Error bars represent standard error ( ns , not significant ; * * , p < 0 . 01 ; two - tailed Student ' s t test ) . Representative Giemsa - stained metaphases of shWRNIP1 cells treated with Mirin alone or in combination with HU . Arrows indicate chromosomal aberrations .

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(E) Experimental scheme of the Mirin aberration analysis is given. The experiment was carried out as in (D) but cells were pre-treated or not with 50 µM Mirin. Dot plot shows the effect of Mirin exposure on the number of chromosome aberrations per cell in shWRNIP1 cells. Horizontal black lines represent the mean SE. Error bars represent standard error (ns, not significant; **, p < 0.01; two-tailed Student's t test). Representative Giemsa-stained metaphases of shWRNIP1 cells treated with Mirin alone or in combination with HU. Arrows indicate chromosomal aberrations.


( F ) Experimental design of the chromosomal aberration assay is reported . shWRNIP1 cells were transfected with control siRNAs ( siCtrl ) or FBH1 siRNA ( siFBH1 ) . Fourth - eight hours thereafter , cells were treated or not with 4 mM HU and then left to recover for 16 h . Metaphases were collected with colcemid and prepared as reported in " Supplemental information " section . Dot plot shows the number of chromosomal aberrations per cell . Western blot shows FBH1 depletion in the cells . The membrane was probed with an anti - FBH1 . GAPDH was used as a loading control . Horizontal black lines represent the mean SE . Error bars represent standard error . ( * * , p < 0 . 01 ; two - tailed Student ' s t test ) .

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(F) Experimental design of the chromosomal aberration assay is reported. shWRNIP1 cells were transfected with control siRNAs (siCtrl) or FBH1 siRNA (siFBH1). Fourth-eight hours thereafter, cells were treated or not with 4 mM HU and then left to recover for 16 h. Metaphases were collected with colcemid and prepared as reported in "Supplemental information" section. Dot plot shows the number of chromosomal aberrations per cell. Western blot shows FBH1 depletion in the cells. The membrane was probed with an anti-FBH1. GAPDH was used as a loading control. Horizontal black lines represent the mean SE. Error bars represent standard error. (**, p < 0.01; two-tailed Student's t test).


SILAC - based proteomics data identify known ( red ) & novel ( green ) hypoxia - induced proteins in SW620 cells . One sample T - test was performed .

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SILAC-based proteomics data identify known (red) & novel (green) hypoxia-induced proteins in SW620 cells. One sample T-test was performed.


Western blotting confirmed GPRC5A as a hypoxia - induced protein in SILAC lysates .

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Western blotting confirmed GPRC5A as a hypoxia-induced protein in SILAC lysates.


Validation of GPRC5A western blot data using siRNA . * Non - specific band of ~ 60kDa not depleted by GPRC5A siRNA .

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Validation of GPRC5A western blot data using siRNA. *Non-specific band of ~60kDa not depleted by GPRC5A siRNA.


Confocal microscopy showing plasma membrane GPRC5A expression in hypoxic SW620 cells ( scale bars : 75um ) .

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Confocal microscopy showing plasma membrane GPRC5A expression in hypoxic SW620 cells (scale bars: 75um).


Western blotting showing GPRC5A upregulation by hypoxia in a panel of colorectal tumour cell lines .

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Western blotting showing GPRC5A upregulation by hypoxia in a panel of colorectal tumour cell lines.


Basal & hypoxia - induced GPRC5A protein expression was decreased by HIF - 1 / 2α depletion .

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Basal & hypoxia-induced GPRC5A protein expression was decreased by HIF-1/2α depletion.


Depletion of HIF - 1β decreased GPRC5A protein upregulation in hypoxia .

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Depletion of HIF-1β decreased GPRC5A protein upregulation in hypoxia.


Hypoxia mimetic DMOG induced HIF - 1 / 2α , CA9 & GPRC5A protein expression . Dual HIF - 1 / 2α depletion reduced GPRC5A induction by DMOG .

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Hypoxia mimetic DMOG induced HIF-1/2α, CA9 & GPRC5A protein expression. Dual HIF-1/2α depletion reduced GPRC5A induction by DMOG.


qRT - PCR demonstrating that GPRC5A mRNA was upregulated by hypoxia . GPRC5A was normalised to HPRT ( error bars ± SD ) .

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qRT-PCR demonstrating that GPRC5A mRNA was upregulated by hypoxia. GPRC5A was normalised to HPRT (error bars ± SD).


qRT - PCR demonstrating that HIF - 1 / 2α depletion decreased GPRC5A induction during hypoxia . GPRC5A was normalised to HPRT ( error bars ± SD ) .

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qRT-PCR demonstrating that HIF-1/2α depletion decreased GPRC5A induction during hypoxia. GPRC5A was normalised to HPRT (error bars ± SD).


ChIP / PCR analyses identify HIF - 1α a binding to the GPRC5A promoter region containing a putative optimal HRE ( error bars ± SD ) .

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ChIP/PCR analyses identify HIF-1α a binding to the GPRC5A promoter region containing a putative optimal HRE (error bars ± SD).


Expression of CA9 in formalin fixed paraffin embedded hypoxic SW620 cells by IHC . Reduced CA9 expression with siRNA confirms antibody specificity ( scale bars : 200um ) .

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Expression of CA9 in formalin fixed paraffin embedded hypoxic SW620 cells by IHC. Reduced CA9 expression with siRNA confirms antibody specificity (scale bars: 200um).


Expression of ; GPRC5A in formalin fixed paraffin embedded hypoxic SW620 cells by IHC . Reduced ; GPRC5A expression with siRNA confirms antibody specificity ( scale bars : 200um ) .

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Expression of ; GPRC5A in formalin fixed paraffin embedded hypoxic SW620 cells by IHC. Reduced ; GPRC5A expression with siRNA confirms antibody specificity (scale bars: 200um).


IHC analysis of serial sections from human colorectal tissue from patients with mesenteric ischaemia ( strangulated colon ) . GPRC5A is co - expressed with CA9 in the colonic epithelial cells ( scale bars : 50um ) .

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IHC analysis of serial sections from human colorectal tissue from patients with mesenteric ischaemia (strangulated colon). GPRC5A is co-expressed with CA9 in the colonic epithelial cells (scale bars: 50um).


Quantitative RT - PCR analysis of mouse intestinal tissue . Gene expression was normalised to housekeeping gene Tbp . Raw data from three independent experiments ( n = 3 mice ) are shown ( error bars ± SEM ) .

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Quantitative RT-PCR analysis of mouse intestinal tissue. Gene expression was normalised to housekeeping gene Tbp. Raw data from three independent experiments (n=3 mice) are shown (error bars ± SEM).


Tg [ fli1 : eGFP ; vhl - / - ] & Tg [ fli1 : eGFP ] zebrafish embryos ( 5 days post - fertilisation ) demonstrate excessive angiogenesis & increased expression of HIF target genes ( scale bars : 100um ) .

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Tg[fli1:eGFP; vhl-/-] & Tg[fli1:eGFP] zebrafish embryos (5 days post-fertilisation) demonstrate excessive angiogenesis & increased expression of HIF target genes (scale bars: 100um).


gprc5ba was induced in vhl mutant zebrafish embryos & fli1 : eGFP zebrafish embryos exposed to 5 % O2 ( vs normoxia ) for 24 hours ( RT - PCR ) .

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gprc5ba was induced in vhl mutant zebrafish embryos & fli1:eGFP zebrafish embryos exposed to 5% O2 (vs normoxia) for 24 hours (RT-PCR).


Kaplan - Meier curve following analysis of transcriptomics dataset GSE24551 . Event - free survival is significantly reduced in patients with tumours expressing high levels of GPRC5A mRNA .

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Kaplan-Meier curve following analysis of transcriptomics dataset GSE24551. Event-free survival is significantly reduced in patients with tumours expressing high levels of GPRC5A mRNA.


GPRC5A depletion markedly increases caspase - 3 activation / PARP cleavage during hypoxia .

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GPRC5A depletion markedly increases caspase-3 activation/PARP cleavage during hypoxia.


Three independent siRNA sequences targeting GPRC5A induce caspase - 3 activation / PARP cleavage during hypoxia .

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Three independent siRNA sequences targeting GPRC5A induce caspase-3 activation/PARP cleavage during hypoxia.


GPRC5A depletion reduces hypoxic cell growth / survival . Crystal violet cell assays show reduced cell growth / survival in GPRC5A depleted cells during hypoxia [ n = 3 ] .

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GPRC5A depletion reduces hypoxic cell growth/survival. Crystal violet cell assays show reduced cell growth/survival in GPRC5A depleted cells during hypoxia [n=3].


Expression of an siRNA - resistant GPRC5A cDNA rescues hypoxic GPRC5A - depleted cells from apoptosis . Upper : doxycycline - induced expression GPRC5Asi1R rescues increases caspase - 3 / PARP cleavage induced by GPRC5A depletion in hypoxia . Lower : generation of an siRNA1 resistant GPRC5A cDNA by synonymous mutations .

O O O O O O B-GENEPROD O O O B-GENEPROD O O O O B-EXP_ASSAY O O O B-SMALL_MOLECULE O O O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O

Expression of an siRNA-resistant GPRC5A cDNA rescues hypoxic GPRC5A-depleted cells from apoptosis. Upper: doxycycline-induced expression GPRC5Asi1R rescues increases caspase-3/PARP cleavage induced by GPRC5A depletion in hypoxia. Lower: generation of an siRNA1 resistant GPRC5A cDNA by synonymous mutations.


Caspase inhibitor QVD prevents caspase - 3 activation / PARP cleavage by GPRC5A depletion in hypoxia .

B-GENEPROD O B-SMALL_MOLECULE O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O B-GENEPROD O O O O

Caspase inhibitor QVD prevents caspase-3 activation/PARP cleavage by GPRC5A depletion in hypoxia.


GPRC5A depletion in hypoxia induces apoptosis as determined by the violet ratiometric membrane asymmetry probe / dead cell apoptosis assay and flow

B-GENEPROD O O O O B-EXP_ASSAY O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY

GPRC5A depletion in hypoxia induces apoptosis as determined by the violet ratiometric membrane asymmetry probe/dead cell apoptosis assay and flow cytometry


Hypoxia induced YAP stabilisation via Ser397 dephosphorylated was abrogated in GPRC5A depleted cells .

O O B-GENEPROD O O O O O O O B-GENEPROD O O O

Hypoxia induced YAP stabilisation via Ser397 dephosphorylated was abrogated in GPRC5A depleted cells.


Hypoxia - induced nuclear localisation of YAP was attenuated in GPRC5A depleted cells .

O O O B-SUBCELLULAR B-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD O O O

Hypoxia-induced nuclear localisation of YAP was attenuated in GPRC5A depleted cells.


Hypoxia activated TEAD activity ( 8x GTIIC - luc reporter ) but this was reduced by GPRC5A depletion . A representative triplicate experiment is shown ( n = 3 ) .

O O B-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD O O O O O O O O O O O O O O

Hypoxia activated TEAD activity (8x GTIIC-luc reporter) but this was reduced by GPRC5A depletion. A representative triplicate experiment is shown (n=3).


Hypoxia reduced LATS activity and expression but this was prevented by GPRC5A depletion .

O O O O O B-EXP_ASSAY O O O O O B-GENEPROD O O

Hypoxia reduced LATS activity and expression but this was prevented by GPRC5A depletion.


Constitutively active RhoA ( G14V ) expression restored YAP stabilisation ( Ser397 dephosphorylation ) by hypoxia in GPRC5A depleted cells .

O O B-GENEPROD O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O B-GENEPROD O O O

Constitutively active RhoA (G14V) expression restored YAP stabilisation (Ser397 dephosphorylation) by hypoxia in GPRC5A depleted cells.


The YAP / TEAD inhibitor verteporfin selectively inhibited cancer cell survival in hypoxia by crystal violet assay ( n = 3 independent experiments ) .

O B-GENEPROD O B-GENEPROD O B-SMALL_MOLECULE O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O

The YAP/TEAD inhibitor verteporfin selectively inhibited cancer cell survival in hypoxia by crystal violet assay (n=3 independent experiments).