Figure 3 ( A ) In vitro phosphorylated GST H2B by Syk kinase was incubated with bacterially purified recombinant wild type SHP - 1 or elution buffer and the amount of released phosphate was assayed colorimetrically using the malachite green reagent ( A620 nm ) . Data represent mean absorbance from four independent experiments . Error bar indicates SD ; * p < 0 . 05 , by Student ' s t test . ( B ) Bacterially purified GST H2B was subjected to in vitro kinase assay using HEK293T cell lysate , and then phosphorylated proteins were incubated with either bacterially purified MBP tagged constitutively active ( SHP - 1 ΔSH2 ) , or catalytically inactive SHP - 1 C / S mutant . The phosphorylation status of recombinant H2B was detected by immunoblotting with phospho - tyrosine antibody . ( C ) Phospho ( pH2B ) Y121 and non - phospho peptide of H2B was incubated with constitutively active ( SHP - 1 ΔSH2 ) or catalytically inactive mutant ( C / S ) of SHP - 1 and the released phosphate was assayed colorimetrically using the malachite green reagent ( A620nm ) . n = 3 independent experiments , Error bars indicate SD ; * * * p < 0 . 001 ( one - way ANOVA , Tukey ' s multiple comparison test ) . ( D ) 293T cells were transfected with vector , Myc tagged SHP - 1 WT or C / S mutant . The phosphorylation status of endogenous H2B was detected by immunoblotting with pY121 - H2B antibody . ( E ) Nuclear extracts of 293T WT and SHP - 1 KO cells were made and the levels of H2B phosphorylation were detected using pY121 - H2B antibody . ( F ) Wild type ( WT ) along with SHP - 1 KO 293T cells transfected with either Myc vector , Myc SHP - 1 or Myc SHP - 1 C / S mutant were lysed . Levels of pY121 - H2B and H2BUb were determined using specific antibodies . ( G ) 293T Cells were treated with SHP - 1 inhibitor TPI ( 50nm ) for 1hr and levels of pY121 - H2B was determined using pY121H2B antibody . MCF7 cells were transduced with ( H ) CTR9 shRNA along with control shRNA . Post 72 h , cells were collected and lysed to isolate soluble and nuclear fractions . Levels of phosphorylated and ubiquitinated H2B were detected by immunoblotting with indicated specific antibodies . MCF7 cells were transduced with ( I ) Paf1 shRNA , along with control shRNA . Post 72 h , cells were collected and lysed to isolate soluble and nuclear fractions . Levels of phosphorylated and ubiquitinated H2B were detected by immunoblotting with indicated specific antibodies . MCF7 cells were transduced with ( J ) RTF1 shRNA along with control shRNA . Post 72 h , cells were collected and lysed to isolate soluble and nuclear fractions . Levels of phosphorylated and ubiquitinated H2B were detected by immunoblotting with indicated specific antibodies .

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

Figure 3(A) In vitro phosphorylated GST H2B by Syk kinase was incubated with bacterially purified recombinant wild type SHP-1 or elution buffer and the amount of released phosphate was assayed colorimetrically using the malachite green reagent (A620 nm). Data represent mean absorbance from four independent experiments. Error bar indicates SD; *p<0.05, by Student's t test.(B) Bacterially purified GST H2B was subjected to in vitro kinase assay using HEK293T cell lysate, and then phosphorylated proteins were incubated with either bacterially purified MBP tagged constitutively active (SHP-1 ΔSH2), or catalytically inactive SHP-1 C/S mutant. The phosphorylation status of recombinant H2B was detected by immunoblotting with phospho-tyrosine antibody.(C) Phospho (pH2B) Y121 and non-phospho peptide of H2B was incubated with constitutively active (SHP-1 ΔSH2) or catalytically inactive mutant (C/S) of SHP-1 and the released phosphate was assayed colorimetrically using the malachite green reagent (A620nm). n=3 independent experiments, Error bars indicate SD; ***p<0.001 (one-way ANOVA, Tukey's multiple comparison test).(D) 293T cells were transfected with vector, Myc tagged SHP-1 WT or C/S mutant. The phosphorylation status of endogenous H2B was detected by immunoblotting with pY121-H2B antibody.(E) Nuclear extracts of 293T WT and SHP-1 KO cells were made and the levels of H2B phosphorylation were detected using pY121-H2B antibody.(F) Wild type (WT) along with SHP-1 KO 293T cells transfected with either Myc vector, Myc SHP-1 or Myc SHP-1 C/S mutant were lysed. Levels of pY121-H2B and H2BUb were determined using specific antibodies.(G) 293T Cells were treated with SHP-1 inhibitor TPI (50nm) for 1hr and levels of pY121-H2B was determined using pY121H2B antibody.MCF7 cells were transduced with (H) CTR9 shRNA along with control shRNA. Post 72 h, cells were collected and lysed to isolate soluble and nuclear fractions. Levels of phosphorylated and ubiquitinated H2B were detected by immunoblotting with indicated specific antibodies.MCF7 cells were transduced with (I) Paf1 shRNA, along with control shRNA. Post 72 h, cells were collected and lysed to isolate soluble and nuclear fractions. Levels of phosphorylated and ubiquitinated H2B were detected by immunoblotting with indicated specific antibodies.MCF7 cells were transduced with (J) RTF1 shRNA along with control shRNA. Post 72 h, cells were collected and lysed to isolate soluble and nuclear fractions. Levels of phosphorylated and ubiquitinated H2B were detected by immunoblotting with indicated specific antibodies.


Figure 4 ( A ) Cell lysates derived from 293T cells transfected with empty vector , SFB - tagged H2B , H2B Y121F or H2B Y121D constructs were pulled down with S - protein agarose beads . RNF20 interaction with H2B was detected by immunoblotting with specific antibody . ( B ) HEK293T cells were co - transfected with the SFB - tagged H2B , H2B Y121F or H2B Y121D constructs along with Myc - tagged UBE2A . Cell lysates were pulled down with S - protein beads , and interaction was detected by immunoblotting with Myc antibody . ( C ) Glutathione Sepharose beads bound with bacterially expressed recombinant GST - H2B , GST H2B Y121F and GST - H2B Y121D proteins were incubated with bacterially purified recombinant His UBE2A , and interaction of UBE2A with H2B was detected by immunoblotting with His antibody . Recombinant protein expression and pulldown was shown by Coomassie staining . ( D ) Bacterially purified GST , GST H2B and GST H2B - p ( phosphorylated using Syk ) was incubated with recombinant His UBE2A , and interaction of UBE2A with H2B was detected by immunoblotting with His antibody . Recombinant protein expression and pulldown was shown by Coomassie staining . ( E ) WT 293T cells along with SHP - 1 KO were co - transfected with SFB vector or SFB H2B along with Myc UbE2A . Cell lysates were pulldown using S - protein beads , and interaction of UBE2A with H2B was determined by Western blotting with the indicated antibodies . ( F ) HEK293T cells were transfected with SFB vector or SFB H2B along with Myc UBE2A . Cells were treated with SHP - 1 inhibitor TPI ( 50nm ) for 1hr after 24hrs of transfection . Cells lysates were subjected to pull - down with S - protein beads , and western blotting was performed using specific antibodies .

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

Figure 4(A) Cell lysates derived from 293T cells transfected with empty vector, SFB-tagged H2B, H2B Y121F or H2B Y121D constructs were pulled down with S-protein agarose beads. RNF20 interaction with H2B was detected by immunoblotting with specific antibody.(B) HEK293T cells were co-transfected with the SFB-tagged H2B, H2B Y121F or H2B Y121D constructs along with Myc-tagged UBE2A. Cell lysates were pulled down with S-protein beads, and interaction was detected by immunoblotting with Myc antibody.(C) Glutathione Sepharose beads bound with bacterially expressed recombinant GST-H2B, GST H2B Y121F and GST-H2B Y121D proteins were incubated with bacterially purified recombinant His UBE2A, and interaction of UBE2A with H2B was detected by immunoblotting with His antibody. Recombinant protein expression and pulldown was shown by Coomassie staining.(D) Bacterially purified GST, GST H2B and GST H2B-p (phosphorylated using Syk) was incubated with recombinant His UBE2A, and interaction of UBE2A with H2B was detected by immunoblotting with His antibody. Recombinant protein expression and pulldown was shown by Coomassie staining.(E) WT 293T cells along with SHP-1 KO were co-transfected with SFB vector or SFB H2B along with Myc UbE2A. Cell lysates were pulldown using S-protein beads, and interaction of UBE2A with H2B was determined by Western blotting with the indicated antibodies.(F) HEK293T cells were transfected with SFB vector or SFB H2B along with Myc UBE2A.Cells were treated with SHP-1 inhibitor TPI (50nm) for 1hr after 24hrs of transfection. Cells lysates were subjected to pull-down with S-protein beads, and western blotting was performed using specific antibodies.


Figure 5 ( A ) Representative super - resolution images of MCF7 cells immunostained with SHP - 1 in combination with CDK9 , total RNA Polymerse II , RNA polymerase II Ser2P , and RNA Polymerase II Ser5P . Scale bar : 5µm . Fluorescence intensities along the line drawn in a representative section of cell were obtained using Zen software and the co - localization plots were made using GraphPad . ( B ) Co - localization of SHP - 1 with CDK9 , total RNA Polymerase II , RNA Polymerase II S2P , and RNA Polymerase II S5P was measured by Pearson correlation coefficient using ImageJ . Values presented as the mean ± SD , n = 10 ( from three independent experiments ) . * * * p value < 0 . 001 . ( One - way ANOVA ) . ( C ) Endogenous interaction of SHP - 1 with RNA Polymerase II , RNA polymerase II Ser2P , and RNA Polymerase II Ser5P and CDK9 was detected by immunoblotting after performing immunoprecipitation with either IgG or SHP - 1 antibody using TurboNuclease treated cell lysate . ( D ) Nuclear extracts of MCF - 7 WT and SHP - 1 knockout cells were made and the levels of RNA Polymerase II , RNA Polymerase II Ser2P and RNA Polymerase II Ser5P were detected by immunoblotting using specific antibodies . ( E ) Nuclear extracts of cells treated with either DMSO or SHP - 1 inhibitor TPI ( 50nm ) were used to detect total RNA Polymerase II and its modifications by immunoblotting using specific antibodies . ( F ) Nuclear extracts of MCF - 7 WT and SHP - 1 knockout cells transfected with Myc vector , H2B Y121F or H2B Y121D constructs were made and the levels of RNA Polymerase II , RNA Polymerase II Ser2P , RNA Polymerase II Ser5P and Lamin B1 were detected by immunoblotting using specific antibodies . ( G ) Immunofluorescence of WT or SHP - 1 knockout MCF7 cells labelled with the 5 - Ethynyl Uridine ( EU ) for 60 minutes to capture nascent RNA synthesis . Scale bars 10μm . ( H ) The EU incorporation in MCF - 7 WT and SHP - 1 KO cells was quantified in three independent experiments . Values presented as the mean ± SD , * * * indicates p < 0 . 001 ( One - way ANOVA ) . ( I ) Representative images of immunofluorescence of DMSO or TPI treated ( 50nm ) MCF7 cells labelled with EU for 60 minutes was shown . Scale bar : 40µm . ( J ) The EU incorporation in MCF7 cells treated with DMSO or TPI was quantified in three independent experiments . Values presented as the mean ± SD , * * * indicates p < 0 . 001 ( One - way ANOVA ) .

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

Figure 5(A) Representative super-resolution images of MCF7 cells immunostained with SHP-1 in combination with CDK9, total RNA Polymerse II, RNA polymerase II Ser2P, and RNA Polymerase II Ser5P. Scale bar: 5µm. Fluorescence intensities along the line drawn in a representative section of cell were obtained using Zen software and the co-localization plots were made using GraphPad.(B) Co-localization of SHP-1 with CDK9, total RNA Polymerase II, RNA Polymerase II S2P, and RNA Polymerase II S5P was measured by Pearson correlation coefficient using ImageJ. Values presented as the mean ± SD, n=10 (from three independent experiments). ***p value < 0.001. (One-way ANOVA).(C) Endogenous interaction of SHP-1 with RNA Polymerase II, RNA polymerase II Ser2P, and RNA Polymerase II Ser5P and CDK9 was detected by immunoblotting after performing immunoprecipitation with either IgG or SHP-1 antibody using TurboNuclease treated cell lysate.(D) Nuclear extracts of MCF-7 WT and SHP-1 knockout cells were made and the levels of RNA Polymerase II, RNA Polymerase II Ser2P and RNA Polymerase II Ser5P were detected by immunoblotting using specific antibodies.(E) Nuclear extracts of cells treated with either DMSO or SHP-1 inhibitor TPI (50nm) were used to detect total RNA Polymerase II and its modifications by immunoblotting using specific antibodies.(F) Nuclear extracts of MCF-7 WT and SHP-1 knockout cells transfected with Myc vector, H2B Y121F or H2B Y121D constructs were made and the levels of RNA Polymerase II, RNA Polymerase II Ser2P, RNA Polymerase II Ser5P and Lamin B1 were detected by immunoblotting using specific antibodies.(G) Immunofluorescence of WT or SHP-1 knockout MCF7 cells labelled with the 5-Ethynyl Uridine (EU) for 60 minutes to capture nascent RNA synthesis. Scale bars 10μm. (H) The EU incorporation in MCF-7 WT and SHP-1 KO cells was quantified in three independent experiments. Values presented as the mean ± SD, ***indicates p<0.001 (One-way ANOVA).(I) Representative images of immunofluorescence of DMSO or TPI treated (50nm) MCF7 cells labelled with EU for 60 minutes was shown. Scale bar: 40µm. (J) The EU incorporation in MCF7 cells treated with DMSO or TPI was quantified in three independent experiments. Values presented as the mean ± SD, ***indicates p<0.001 (One-way ANOVA).


Figure 7 ( C , D ) MCF - 7 WT cells along with SHP - 1 KO cells transfected with either vector , H2B Y121F or H2B Y121D mutant were used for ChIP q - PCR analysis of the H2BUb at indicated genes . The data shown is derived from two independent experiments . Error bars indicate the mean ± SD .

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

Figure 7(C, D) MCF-7 WT cells along with SHP-1 KO cells transfected with either vector, H2B Y121F or H2B Y121D mutant were used for ChIP q-PCR analysis of the H2BUb at indicated genes. The data shown is derived from two independent experiments. Error bars indicate the mean ± SD.


Figure 8 ( A ) Lysates of WT and SHP - 1 knockout MCF7 cells were made and the levels of LC3 - I and II were detected by immunoblotting . ( B ) Levels of LC3 II were normalized against total LC3 ( LC3 I + LC3 II ) . The data shown is derived from three independent experiments . Error bars indicate the mean ± SD . * p value < 0 . 05 , Student ' s t - test . ( C ) Representative super - resolution images of WT and SHP - 1 KO MCF7 cells immunostained with LC3 antibody , ( Scale bar : 5µm ) . ( D ) Quantification of LC3 puncta accumulation per cell done by using ImageJ was shown . Bars represents mean ± SD . ( N = 60 ) * * * p value < 0 . 001 , Student ' s t - test . ( E ) WT along with SHP - 1 KO MCF7 cells transfected with either Myc vector , Myc SHP - 1 or Myc SHP - 1 NLS mutant were lysed . Levels of LC3 and actin were determined using specific antibodies . ( F ) WT and SHP - 1 knockout MCF7 cells transfected with the Myc vector or Myc - tagged H2B , H2B Y121F or H2B Y121D constructs were immunostained with endogenous p62 and Myc antibody . Image acquisition was done using LSM 700 , ( Scale bar : 20µm ) . ( G ) Accumulation of p62 was quantified by counting p62 positive puncta per cell . ( N = 25 ) ; values presented as the mean ± SD . * * * p value < 0 . 001 ( One - way ANOVA ) . ( H ) Representative super - resolution images of LC3 and LAMP2 immunostaining in WT and SHP - 1 KO MCF7 cells . ( Scale bar : 20 µm for merge & 5 µm for Zoom ) . ( I ) LC3 puncta co - localizing with LAMP2 per cell were quantified using ImageJ . ( N = 45 ) ; values presented as the mean ± SD . * * * p value < 0 . 001 , Student ' s t - test .

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

Figure 8(A) Lysates of WT and SHP-1 knockout MCF7 cells were made and the levels of LC3-I and II were detected by immunoblotting. (B) Levels of LC3 II were normalized against total LC3 (LC3 I+LC3 II). The data shown is derived from three independent experiments. Error bars indicate the mean ± SD. *p value < 0.05, Student's t-test.(C) Representative super- resolution images of WT and SHP-1 KO MCF7 cells immunostained with LC3 antibody, (Scale bar: 5µm). (D) Quantification of LC3 puncta accumulation per cell done by using ImageJ was shown. Bars represents mean ± SD.(N=60) ***p value < 0.001, Student's t-test.(E) WT along with SHP-1 KO MCF7 cells transfected with either Myc vector, Myc SHP-1 or Myc SHP-1 NLS mutant were lysed. Levels of LC3 and actin were determined using specific antibodies.(F) WT and SHP-1 knockout MCF7 cells transfected with the Myc vector or Myc-tagged H2B, H2B Y121F or H2B Y121D constructs were immunostained with endogenous p62 and Myc antibody. Image acquisition was done using LSM 700, (Scale bar: 20µm). (G) Accumulation of p62 was quantified by counting p62 positive puncta per cell. (N= 25); values presented as the mean ± SD. ***p value < 0.001 (One-way ANOVA).(H) Representative super-resolution images of LC3 and LAMP2 immunostaining in WT and SHP-1 KO MCF7 cells. (Scale bar: 20 µm for merge & 5 µm for Zoom). (I) LC3 puncta co-localizing with LAMP2 per cell were quantified using ImageJ. (N=45); values presented as the mean ± SD. ***p value < 0.001, Student's t-test.


Figure 1 B Chromatin accessibility as measured by the number of all ( TSS and distal ) ATAC - Seq peaks ( read count ≥ 10 ) detected in DN2 ( n = 5 ) , DN3 ( n = 5 ) , ISP ( n = 5 ) , DPCD3 - ( n = 6 ) , DPCD3 + ( n = 6 ) , SPCD4 + ( n = 5 ) , SPCD8 + ( n = 5 ) populations . Horizontal lines of the box plot indicate the median , lower and upper limits of each box correspond to the first and third quartiles ( the 25th and 75th percentiles ) and the lower and upper whiskers extend from min to max ; pval = 0 . 035 ( Kruskal - Wallis Test ) . C Fraction of OCRs having increasing , decreasing , steady and fluctuating accessibility pattern during T - cell development ( x - axis ) . Average number of normalized reads in OCRs ( y - axis ) .

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

Figure 1 B Chromatin accessibility as measured by the number of all (TSS and distal) ATAC-Seq peaks (read count ≥10) detected in DN2 (n=5), DN3 (n=5), ISP (n=5), DPCD3- (n=6), DPCD3+ (n=6), SPCD4+ (n=5), SPCD8+ (n=5) populations. Horizontal lines of the box plot indicate the median, lower and upper limits of each box correspond to the first and third quartiles (the 25th and 75th percentiles) and the lower and upper whiskers extend from min to max; pval=0.035 (Kruskal-Wallis Test). C Fraction of OCRs having increasing, decreasing, steady and fluctuating accessibility pattern during T-cell development (x-axis). Average number of normalized reads in OCRs (y-axis).


Figure 2 A Unsupervised hierarchical clustering based on the variance stabilizing transformation ( VST ) normalized read counts ( each row is also normalized by the row mean ) of the 2 , 021 signature open chromatin regions of 7 stages B ATAC - Seq genome tracks showing three signature peaks located near the genes SAE1 , RAG1 , and CD8A . Framed regions indicate cell - type - specific signature ATAC - Seq peaks . Blue triangles indicate known promoter or enhancer regions , which are annotated in the GeneHancer ( GH ) Regulatory Elements database

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

Figure 2 A Unsupervised hierarchical clustering based on the variance stabilizing transformation (VST) normalized read counts (each row is also normalized by the row mean) of the 2,021 signature open chromatin regions of 7 stages  B ATAC-Seq genome tracks showing three signature peaks located near the genes SAE1, RAG1, and CD8A. Framed regions indicate cell-type-specific signature ATAC-Seq peaks. Blue triangles indicate known promoter or enhancer regions, which are annotated in the GeneHancer (GH) Regulatory Elements database


Figure 3A The rationale of footprinting analysis as illustrated by actual ATAC - Seq peaks , HINT - ATAC footprint hits , and example TF motif instances near RAG1 gene in DN2 population of thymus donor 6 . B Heatmap of ln ( odds ratio ) of the 50 TFs showing the most differential abundance of binding motifs in the open chromatin regions . TFs with an SD of the motif counts < 10 are excluded .

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

Figure 3A The rationale of footprinting analysis as illustrated by actual ATAC-Seq peaks, HINT-ATAC footprint hits, and example TF motif instances near RAG1 gene in DN2 population of thymus donor 6.B Heatmap of ln(odds ratio) of the 50 TFs showing the most differential abundance of binding motifs in the open chromatin regions. TFs with an SD of the motif counts <10 are excluded.


Figure 4 D Chromatin accessibility as assessed by the number of ATAC peaks identified in the sorted populations of normal thymic T - cell precursors and in T - ALLs , respectively ( n = 10 / 19 patients have two biological replicates , n = 9 / 19 patients have one replicate ) . Horizontal lines of the box plot indicate the median , lower and upper limits of each box correspond to the first and third quartiles ( the 25th and 75th percentiles ) and the lower and upper whiskers extend from min to max ; pval = 4 . 4e - 08 ( Kruskal - Wallis Test ) . E Heatmap representing the enrichment and depletion of binding motifs of top 50 TFs in OCRs of T - ALLs in comparison to healthy T - cell precursors . Patients on the x - axis ; same order as in panel ( C ) . Gray cells represent TFs having non - significant enrichment or depletion .

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

Figure 4 D Chromatin accessibility as assessed by the number of ATAC peaks identified in the sorted populations of normal thymic T-cell precursors and in T-ALLs, respectively (n=10/19 patients have two biological replicates, n=9/19 patients have one replicate). Horizontal lines of the box plot indicate the median, lower and upper limits of each box correspond to the first and third quartiles (the 25th and 75th percentiles) and the lower and upper whiskers extend from min to max; pval=4.4e-08 (Kruskal-Wallis Test).  E Heatmap representing the enrichment and depletion of binding motifs of top 50 TFs in OCRs of T-ALLs in comparison to healthy T-cell precursors. Patients on the x-axis; same order as in panel (C). Gray cells represent TFs having non-significant enrichment or depletion.


Figure 5 C log2 fold changes of 292 genes which are differentially expressed ( x - axis , RNA - Seq : non - sorted bulk thymi vs . T - ALLs ) and differentially accessible ( y - axis , ATAC - Seq : sorted T - cell populations vs . T - ALLs ) in T - ALLs in comparison to healthy T - cell precursors . Dot size and color indicate the frequency ( number of patients having dysregulation of the gene ) . Genes with ≥ 5 / 19 frequency are shown on the plot . Protein coding genes with ≥ 12 / 19 frequency are labelled . For plotting the average LFC values of ATAC and RNA are used per gene per quartile . Q1 - 4 stands for the quadrant 1 - 4 . D Heatmap of VST normalized read counts ( each row is also normalized by the row mean ) of differentially expressed genes ( pval < 0 . 1 ; DESeq2 ) in three patients in comparison to remaining 16 . Orange triangles indicate three patients with no DAB1 but high SPI1 expression and motif counts .

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

Figure 5 C log2 fold changes of 292 genes which are differentially expressed (x-axis, RNA-Seq: non-sorted bulk thymi vs. T-ALLs) and differentially accessible (y-axis, ATAC-Seq: sorted T-cell populations vs. T-ALLs) in T-ALLs in comparison to healthy T-cell precursors. Dot size and color indicate the frequency (number of patients having dysregulation of the gene). Genes with ≥5/19 frequency are shown on the plot. Protein coding genes with ≥12/19 frequency are labelled. For plotting the average LFC values of ATAC and RNA are used per gene per quartile. Q1 - 4 stands for the quadrant 1 - 4. D Heatmap of VST normalized read counts (each row is also normalized by the row mean) of differentially expressed genes (pval<0.1; DESeq2) in three patients in comparison to remaining 16. Orange triangles indicate three patients with no DAB1 but high SPI1 expression and motif counts.


Figure 1Levels of differential free amino acids in MERRF patients ' muscle ( B ) by LC - MS analysis , expressed relative to CTL . In panels the dashed line indicates the Mean CTL value set at 1 . In panels B data are presented as Mean ± SEM . Muscle : MERRF ( n = 10 ) , CTL ( n = 15 ) ( H ) Western blots of muscle lysates separated by denaturing SDS - PAGE and probed for OGDH , ALT2 , α - actin , and GOT2 ( top panel ) , ALT1 and GAPDH ( middle panel ) , GOT1 and GAPDH ( bottom panel ) , C ( CTL ) , M ( MERRF ) .

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

Figure 1Levels of differential free amino acids in MERRF patients' muscle (B) by LC-MS analysis, expressed relative to CTL. In panels the dashed line indicates the Mean CTL value set at 1. In panels B data are presented as Mean ± SEM. Muscle: MERRF (n=10), CTL (n=15)(H) Western blots of muscle lysates separated by denaturing SDS-PAGE and probed for OGDH, ALT2, α-actin, and GOT2 (top panel), ALT1 and GAPDH (middle panel), GOT1 and GAPDH (bottom panel), C (CTL), M (MERRF).


Figure 2 ( A - V ) Age - dependent muscle protein levels of ALT2 ( A ) , GOT2 ( C ) , OGDH ( E ) , GOT1 ( G ) , ALT1 ( I ) , AGC1 ( K ) , SUCLA2 ( M ) , BCAT2 ( O ) , BCKDH ( Q ) , PCCA ( S ) , and MUT ( U ) , estimated by band densitometry normalized by GAPDH , in COX10 KO expressed relative to same age CTL set at 1 . Representative western blots of muscle lysates from 170 - 200 days old mice separated by denaturing SDS - PAGE and probed for ALT2 ( B ) , GOT2 ( D ) , OGDH ( F ) , GOT1 ( H ) , ALT1 ( J ) , AGC1 ( L ) , SUCLA2 ( N ) , BCAT2 ( P ) , BCKDHB ( R ) , PCCA ( T ) , and MUT ( V ) and GAPDH ( B , D , F , H , J , L , N , P , R , T , V ) . : In panels data are presented as Mean ± SD . COX10 KO ( n = 4 ) , CTL ( n = 4 ) . Statistically significant differences between the two groups were estimated by unpaired two - tailed Student ' s test . * , p < 0 . 05 * * , p < 0 . 005 COX10 KO vs CTL . ( X ) Muscle fractional incorporation of M + 6 of glutamate , M + 5 of αKG , M + 4 of fumarate , M + 4 of malate , M + 3 of alanine and M + 3 of lactate , in 200 days COX10 KO and CTL mice IP injected with [ 13C5 , 15N ] - glutamate . In panels X data are presented as Mean ± SEM . In panel X , COX10 KO ( n = 4 ) , CTL ( n = 4 ) . Statistically significant differences between the two groups were estimated by unpaired two - tailed Student ' s test . * , p < 0 . 05 * * , p < 0 . 005 COX10 KO vs CTL . ( Z ) Muscle fractional incorporation of M + 6 of valine , M + 5 of α - KIV , M + 4 of β - ΗΙΒ , M + 3 of propionyl carnitine , in 200 days COX10 KO and CTL mice IP injected with [ 13C5 , 15N ] - valine . In panels Z , data are presented as Mean ± SEM . In panel Z , COX10 KO ( n = 3 ) , CTL ( n = 3 ) . Statistically significant differences between the two groups were estimated by unpaired two - tailed Student ' s test . * , p < 0 . 05 * * , p < 0 . 005 COX10 KO vs CTL .

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

Figure 2(A-V) Age-dependent muscle protein levels of ALT2 (A), GOT2 (C), OGDH (E), GOT1 (G), ALT1 (I), AGC1 (K), SUCLA2 (M), BCAT2 (O), BCKDH (Q), PCCA (S), and MUT (U), estimated by band densitometry normalized by GAPDH, in COX10 KO expressed relative to same age CTL set at 1. Representative western blots of muscle lysates from 170-200 days old mice separated by denaturing SDS-PAGE and probed for ALT2 (B), GOT2 (D), OGDH (F), GOT1 (H), ALT1 (J), AGC1 (L), SUCLA2 (N), BCAT2 (P), BCKDHB (R), PCCA (T), and MUT (V) and GAPDH (B, D, F, H, J, L, N, P, R, T, V). : In panels data are presented as Mean ± SD. COX10 KO (n=4), CTL (n=4). Statistically significant differences between the two groups were estimated by unpaired two-tailed Student's test. *, p<0.05 **, p<0.005 COX10 KO vs CTL.(X) Muscle fractional incorporation of M+6 of glutamate, M+5 of αKG, M+4 of fumarate, M+4 of malate, M+3 of alanine and M+3 of lactate, in 200 days COX10 KO and CTL mice IP injected with [13C5, 15N]-glutamate. In panels X data are presented as Mean ± SEM. In panel X, COX10 KO (n=4), CTL (n=4). Statistically significant differences between the two groups were estimated by unpaired two-tailed Student's test. *, p<0.05 **, p<0.005 COX10 KO vs CTL.(Z) Muscle fractional incorporation of M+6 of valine, M+5 of α-KIV, M+4 of β-ΗΙΒ, M+3 of propionyl carnitine, in 200 days COX10 KO and CTL mice IP injected with [13C5, 15N]-valine. In panels Z, data are presented as Mean ± SEM. In panel Z, COX10 KO (n=3), CTL (n=3). Statistically significant differences between the two groups were estimated by unpaired two-tailed Student's test. *, p<0.05 **, p<0.005 COX10 KO vs CTL.


Figure 3 ( B ) Age - dependent Ph - 4EBP1 / 4E - BP1 ratio in COX10 KO expressed relative to same age CTL set at 1 . ( C ) Representative western blot of muscle lysates from 200 days old mice separated by denaturing SDS - PAGE and probed for Ph - 4E - BP1 and for 4E - BP1 . In panels data are presented as Mean ± SD . COX10 KO ( n = 4 ) , CTL ( n = 4 ) . * , p < 0 . 05 COX10 KO vs . same age CTL . ( D ) Age - dependent protein levels of Ph - 4E - BP1 in COX10 KO muscle estimated by band densitometry normalized by GAPDH expressed relative to 50 days COX10 KO set at 1 . ( E ) Western blots of muscle lysates from 50 , 100 , and 200 days old COX10 KO mice , separated by denaturing SDS - PAGE and probed for Ph - 4E - BP1 and GAPDH . In panel D , data are presented as Mean ± SD . # , p < 0 . 05 COX10 KO vs . COX10 KO at different ages . 50d ( n = 3 ) , 100d ( n = 3 ) , 200d ( n = 3 ) . Age - dependent protein levels of SESTRIN 2 ( F ) , REDD1 ( H ) , LC3II ( J ) 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 SESTRIN 2 ( G ) , REDD1 ( I ) , LC3II ( K ) In panels data are presented as Mean ± SD . COX10 KO ( n = 4 ) , CTL ( n = 4 ) . * , p < 0 . 05 COX10 KO vs . same age CTL . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(B) Age-dependent Ph-4EBP1/4E-BP1 ratio in COX10 KO expressed relative to same age CTL set at 1. (C) Representative western blot of muscle lysates from 200 days old mice separated by denaturing SDS-PAGE and probed for Ph-4E-BP1 and for 4E-BP1. In panels data are presented as Mean ± SD. COX10 KO (n=4), CTL (n=4). *, p<0.05 COX10 KO vs. same age CTL.(D) Age-dependent protein levels of Ph-4E-BP1 in COX10 KO muscle estimated by band densitometry normalized by GAPDH expressed relative to 50 days COX10 KO set at 1. (E) Western blots of muscle lysates from 50, 100, and 200 days old COX10 KO mice, separated by denaturing SDS-PAGE and probed for Ph-4E-BP1 and GAPDH. In panel D, data are presented as Mean ± SD. #, p<0.05 COX10 KO vs. COX10 KO at different ages. 50d (n=3), 100d (n=3), 200d (n=3).Age-dependent protein levels of SESTRIN 2 (F), REDD1 (H), LC3II (J) 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 SESTRIN 2 (G), REDD1 (I), LC3II (K) In panels data are presented as Mean ± SD. COX10 KO (n=4), CTL (n=4). *, p<0.05 COX10 KO vs. same age CTL.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.


Figure 4Age - 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 ) . ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4Age-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).(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.


Figure 5 ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(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.


Figure 6Age - 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 . ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6Age-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.(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.


Figure 7 ( 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 ) . ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7(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).(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.


Figure 1 ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(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.


Figure 2 ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(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).


Figure 3 ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(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.


Figure 6 ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(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.


Figure 1 ( 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 . ( 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 ) . ( 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 ) . 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-PANEL_START O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(A) 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.(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). (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).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).


Figure 2 ( 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 ) . ( 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 . ( 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 . ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(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).(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.(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.(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.


Figure 3B ) 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 . 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 . 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 . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3B) 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.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.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.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.


Figure 4A ) 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 . 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 . 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 . ( 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 . ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A) 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.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.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.(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.(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.


Figure 1 ( 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 ) . ( 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 . ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(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).(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.(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.


Figure 2 ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(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.