( E ) Fold increase of Oct4 - GFP + colony number over control under forced expression of individual factors . n ≥ 5 , where each dot indicates an independent experiment . Box - plots show median , 1st and 3rd quartile values . One - sample Wilcoxon test p - values are as indicated , with p values < 0 . 05 shown in

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(E) Fold increase of Oct4-GFP+ colony number over control under forced expression of individual factors. n ≥ 5, where each dot indicates an independent experiment. Box-plots show median, 1st and 3rd quartile values. One-sample Wilcoxon test p-values are as indicated, with p values < 0.05 shown in red


( B ) Heatmap of average gene expression normalised to β - actin over an EpiSC resetting time course in 2i + LIF . Each row is coloured according to the unique minimum and maximum for that gene . The values shown are average expression of four independent

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(B) Heatmap of average gene expression normalised to β-actin over an EpiSC resetting time course in 2i+LIF. Each row is coloured according to the unique minimum and maximum for that gene. The values shown are average expression of four independent experiments


( C ) Gene expression for Stat3 , Klf2 , Esrrb and Tfcp2l1 during EpiSC resetting relative to established mouse ESCs . β - actin serves as an internal control . Mean + / - SEM , n = 4 independent experiments . * = p < 0 . 05 Student " s

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(C) Gene expression for Stat3, Klf2, Esrrb and Tfcp2l1 during EpiSC resetting relative to established mouse ESCs. β-actin serves as an internal control. Mean +/- SEM, n=4 independent experiments. * = p<0.05 Student"s t


( E ) Representative confocal images ( top ) and quantification ( bottom ) of Oct4 - GFP reporter mean intensity . The indicated cell lines were treated with DOX for the first 2 days and imaged at Day 6 . Mean + / - SD of 9 technical replicates . One representative experiment of two is shown . Scale bars =

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(E) Representative confocal images (top) and quantification (bottom) of Oct4-GFP reporter mean intensity. The indicated cell lines were treated with DOX for the first 2 days and imaged at Day 6. Mean +/- SD of 9 technical replicates. One representative experiment of two is shown. Scale bars=300µm


( F ) Representative alkaline phosphatase ( AP ) staining images ( left ) and quantification ( right ) of AP + colonies after clonal replating , as described in

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(F) Representative alkaline phosphatase (AP) staining images (left) and quantification (right) of AP+ colonies after clonal replating, as described in panel


( A ) Top : Predictions of the number of regulation steps required for full activation of the indicated gene under control or dual expression of Esrrb and Tbx3 ( + E / T ) . Bottom : Gene expression of EpiSCs harbouring empty vector ( grey ) or Esrrb / Tbx3 ( orange ) , capture at D0 ( F / A ) , D2 and D4 ( as described in Fig 3D ) . Dashed black line : expression levels in ESCs maintained in 2i + LIF . Data normalised to empty vector cultures in F / A . Gapdh serves as an internal control . Mean + / - SEM , n = 3 independent experiments . ( B ) As for panel A , comparison of control with dual expression of Esrrb and Klf4 ( + E / K , green in bottom plot ) . Mean + / - SEM , n = 3 independent experiments for bottom qRT - PCR

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(A) Top: Predictions of the number of regulation steps required for full activation of the indicated gene under control or dual expression of Esrrb and Tbx3 (+E/T). Bottom: Gene expression of EpiSCs harbouring empty vector (grey) or Esrrb/Tbx3 (orange), capture at D0 (F/A), D2 and D4 (as described in Fig 3D). Dashed black line: expression levels in ESCs maintained in 2i+LIF. Data normalised to empty vector cultures in F/A. Gapdh serves as an internal control. Mean +/- SEM, n=3 independent experiments. (B) As for panel A, comparison of control with dual expression of Esrrb and Klf4 (+E/K, green in bottom plot). Mean +/- SEM, n=3 independent experiments for bottom qRT-PCR panels


( C ) Left : Flow cytometry profiles of the resetting progression of EpiSCs stably transfected with the Esrrb - T2A - Klf4 construct and cultured in 2i + LIF with DOX for 2 and 4 days , with the indicated fraction of cells sorted for colony formation assay . Since the Venus reporter is under the control of a DOX responsive element , and the emission spectra of Venus and GFP fluorescence overlap , Oct4 - GFP reporter could not be fully distinguished from Venus expression . Right : Number of AP + colonies formed from 250 sorted cells from indicated fractions . Data points represent two technical replicates of one out of two independent

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(C) Left: Flow cytometry profiles of the resetting progression of EpiSCs stably transfected with the Esrrb-T2A-Klf4 construct and cultured in 2i+LIF with DOX for 2 and 4 days, with the indicated fraction of cells sorted for colony formation assay. Since the Venus reporter is under the control of a DOX responsive element, and the emission spectra of Venus and GFP fluorescence overlap, Oct4-GFP reporter could not be fully distinguished from Venus expression. Right: Number of AP+ colonies formed from 250 sorted cells from indicated fractions. Data points represent two technical replicates of one out of two independent experiments


( D ) Heatmap of single - cell expression measured by qRT - PCR of major ESC and EpiSC markers in un - induced EpiSCs ( black ) , established ESCs ( red ) , Day 2 High / Low ( dark and light blue ) , and Day 4 High cells ( green )

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(D) Heatmap of single-cell expression measured by qRT-PCR of major ESC and EpiSC markers in un-induced EpiSCs (black), established ESCs (red), Day 2 High/Low (dark and light blue), and Day 4 High cells (green)


( E ) Scatterplots of the relative expression of pluripotency and transitional markers . Red bar corresponds to median gene expression , and each dot represents a single

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(E) Scatterplots of the relative expression of pluripotency and transitional markers. Red bar corresponds to median gene expression, and each dot represents a single cell


Left : 0 . 782 cABN predictions of the sequence of gene activation between gene pairs ( white , OFF ; blue , ON ) along the resetting trajectory , compared to single cell gene expression measured by RT - qPCR . Each table summarises the percentage of single cells at the indicated stage of resetting ( columns ) that have the indicated expression state ( rows ) . Right : Scatterplots showing single cell coordinates based on the expression of the gene

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Left: 0.782 cABN predictions of the sequence of gene activation between gene pairs (white, OFF; blue, ON) along the resetting trajectory, compared to single cell gene expression measured by RT-qPCR. Each table summarises the percentage of single cells at the indicated stage of resetting (columns) that have the indicated expression state (rows). Right: Scatterplots showing single cell coordinates based on the expression of the gene pair


( A ) 0 . 782 cABN predictions of essential or dispensable factors for EpiSC resetting , compared with the experiment results shown in panel ( B ) . ( B ) siRNA knockdown effects measured by Oct4 - GFP + colony formation at Day 6 of resetting . n = 4 independent

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(A) 0.782 cABN predictions of essential or dispensable factors for EpiSC resetting, compared with the experiment results shown in panel (B). (B) siRNA knockdown effects measured by Oct4-GFP+ colony formation at Day 6 of resetting. n=4 independent experiments


( C ) Left , resetting capacity of Klf2 and Klf4 KO EpiSCs measured by Oct4 - GFP + colony formation at Day 6 of resetting . Data points represent two independent experiments . Right , representative fluorescent and bright field images of wild type and Klf2 KO EpiSCs at Day 6 of resetting in 2i +

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(C) Left, resetting capacity of Klf2 and Klf4 KO EpiSCs measured by Oct4-GFP+ colony formation at Day 6 of resetting. Data points represent two independent experiments. Right, representative fluorescent and bright field images of wild type and Klf2 KO EpiSCs at Day 6 of resetting in 2i+LIF


( D ) Expression of naïve pluripotency , transition and somatic lineage markers in wild type and Klf2 KO EpiSCs during a resetting time course in 2i + LIF . Expression is normalised to wild type EpiSCs in A / F , and β - actin was used as internal control . The values shown correspond to the average expression of three technical replicates from one representative experiment out of

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(D) Expression of naïve pluripotency, transition and somatic lineage markers in wild type and Klf2 KO EpiSCs during a resetting time course in 2i+LIF. Expression is normalised to wild type EpiSCs in A/F, and β-actin was used as internal control. The values shown correspond to the average expression of three technical replicates from one representative experiment out of two


( E ) Rescue of Klf2 KO EpiSC resetting by forced expression of individual network components measured by Oct4 - GFP + colony formation at Day 6 of resetting in 2i + LIF . Data points represent two independent

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(E) Rescue of Klf2 KO EpiSC resetting by forced expression of individual network components measured by Oct4-GFP+ colony formation at Day 6 of resetting in 2i+LIF. Data points represent two independent experiments


( F ) Comparison between the effect of single factor knockdowns on ESC maintenance ( n = 4 independent experiments ) and EpiSC resetting using experimental

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(F) Comparison between the effect of single factor knockdowns on ESC maintenance (n=4 independent experiments) and EpiSC resetting using experimental results


( G ) EpiSC resetting in 2i + LIF measured by Oct4 - GFP + colony formation after Stat3 siRNA in wild type EpiSCs ( left ) , or Klf4 KO EpiSCs transfected with Tfcp2l1 and Gbx2 siRNAs . n = 3 independent experiments in wild type cells ( box plots indicate min , median , max ) , n = 2 for Klf4 - / - cells ( box plots indicate min , mean , max )

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(G) EpiSC resetting in 2i+LIF measured by Oct4-GFP+ colony formation after Stat3 siRNA in wild type EpiSCs (left), or Klf4 KO EpiSCs transfected with Tfcp2l1 and Gbx2 siRNAs. n=3 independent experiments in wild type cells (box plots indicate min, median, max), n=2 for Klf4-/- cells (box plots indicate min, mean, max)


( H ) EpiSC resetting in 2i + LIF measured by Oct4 - GFP + colony formation of Stat3 knockdown EpiSCs transiently transfected with Tfcp2l1 , Gbx2 and Klf4 . n = 4 independent experiments : Student " s t - test , p - value indicated on plot . Box plots indicate min , median , max . See also Appendix

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(H) EpiSC resetting in 2i+LIF measured by Oct4-GFP+ colony formation of Stat3 knockdown EpiSCs transiently transfected with Tfcp2l1, Gbx2 and Klf4. n=4 independent experiments: Student"s t-test, p-value indicated on plot. Box plots indicate min, median, max. See also Appendix Fi


( B ) Comparison of predictions ( left ) and experimental outcome ( right ) for the potency of additional network factors in OSKM - driven MEF reprogramming in LIF containing medium n = 4 independent experiments , except Klf2 and Nanog where n = 2 independent experiments . * = p < 0 . 05 Student " s t - test . n . s . = not significant . Red dashed lines indicate empty vector + OSKM ( C ) Comparison of predictions ( left ) and experimental outcome ( right ) for the potency of additional network factors in OSKM - driven MEF reprogramming in 2i + LIF . n = 4 independent experiments , except Klf2 and Nanog where n = 2 independent experiments . * = p < 0 . 05 Student " s t - test . n . s . = not significant . Empty vector + OSKM reprogramming in LIF ( ' control no 2i ' ) was included as a control for the effect of 2i addition . Red dashed lines indicate empty vector + OSKM control level . Box plots indicate min ,

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(B) Comparison of predictions (left) and experimental outcome (right) for the potency of additional network factors in OSKM-driven MEF reprogramming in LIF containing medium n=4 independent experiments, except Klf2 and Nanog where n=2 independent experiments. * = p<0.05 Student"s t-test. n.s. = not significant. Red dashed lines indicate empty vector + OSKM (C) Comparison of predictions (left) and experimental outcome (right) for the potency of additional network factors in OSKM-driven MEF reprogramming in 2i+LIF. n=4 independent experiments, except Klf2 and Nanog where n=2 independent experiments. * = p<0.05 Student"s t-test. n.s. = not significant. Empty vector + OSKM reprogramming in LIF ('control no 2i') was included as a control for the effect of 2i addition. Red dashed lines indicate empty vector + OSKM control level. Box plots indicate min, median


( D ) Recapitulation of the gene activation kinetics during MEF reprogramming . Top , the number of regulation steps required for permanent activation of the indicated gene . Tfcp2l1 and Sall4 are found to activate earlier than Nanog and Esrrb . Bottom , gene expression measured from sorted populations of reprogramming intermediates from O " Malley et al . (

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(D) Recapitulation of the gene activation kinetics during MEF reprogramming. Top, the number of regulation steps required for permanent activation of the indicated gene. Tfcp2l1 and Sall4 are found to activate earlier than Nanog and Esrrb. Bottom, gene expression measured from sorted populations of reprogramming intermediates from O"Malley et al. (


A Heat map from RNA - Seq data from FACS - isolated muscle stem cells and single myofibers showing relative expression of Pax7 , Myf5 , MyoD , Myog and Myf6 .

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A Heat map from RNA-Seq data from FACS-isolated muscle stem cells and single myofibers showing relative expression of Pax7, Myf5, MyoD, Myog and Myf6.


H Colormap of Myf5 , MyoD and Myf6 peaks within 100kb of the Transcription Start Sites ( TSS ) of myokines ( supplemental table EV3 ) ranging from zero ( black ) to six peaks ( red ) occupancy . The onset of differentiation coincides with increased binding of MRFs to the regulatory domains of the myokines genes .

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H Colormap of Myf5, MyoD and Myf6 peaks within 100kb of the Transcription Start Sites (TSS) of myokines (supplemental table EV3) ranging from zero (black) to six peaks (red) occupancy. The onset of differentiation coincides with increased binding of MRFs to the regulatory domains of the myokines genes.


A Gene expression analysis of myokines ( table EV3 ) during a five day time course of myogenic differentiation going from cycling myoblasts in growth media ( Ham ' s F10 supplemented with 20 % Fetal Bovine Serum , 1 % penicillin / streptomycin , 2 . 5 ng / ml basic Fibroblast Growth Factor ) to terminally differentiated myocytes ( 2 days in differentiation media , DMEM supplemented with 5 % horse serum ) to post mitotic multinucleated myotubes ( 5 days in differentiation media ) . Gene expression was assayed in biological triplicate by microarray ( Soleimani et al . , 2018 ; Soleimani et al . , 2012a ) . Expression of each gene was averaged across three replicates and normalized to mean zero and standard deviation of one . Expression values were then truncated at + / - 3 for color display . Blue indicates lower than average expression , while red indicates higher than average expression . B Distribution of Myf6 peaks within 100kb of the TSS of the myokine genes ( table EV3 ) , similar to the analysis shown in ( A ) .

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A Gene expression analysis of myokines (table EV3) during a five day time course of myogenic differentiation going from cycling myoblasts in growth media (Ham's F10 supplemented with 20% Fetal Bovine Serum, 1% penicillin/streptomycin, 2.5 ng/ml basic Fibroblast Growth Factor) to terminally differentiated myocytes (2 days in differentiation media, DMEM supplemented with 5% horse serum) to post mitotic multinucleated myotubes (5 days in differentiation media). Gene expression was assayed in biological triplicate by microarray (Soleimani et al., 2018; Soleimani et al., 2012a). Expression of each gene was averaged across three replicates and normalized to mean zero and standard deviation of one. Expression values were then truncated at +/- 3 for color display. Blue indicates lower than average expression, while red indicates higher than average expression. B Distribution of Myf6 peaks within 100kb of the TSS of the myokine genes (table EV3), similar to the analysis shown in (A).


C - E Snapshots of the UCSC genome browser showing enrichment of Myf6 ChIP - Seq reads compared to control around EGF ( C ) , LIF and OSM ( D ) , and VEGFA ( E ) loci superimposed on reads for Histone H3 lysine 4 mono methyl ( H3K4me1 ) , Histone H3 lysine 4 tri methyl ( H3K4me3 ) and total H3 in myoblasts ( pink ) and myotubes ( green ) .

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C-E Snapshots of the UCSC genome browser showing enrichment of Myf6 ChIP-Seq reads compared to control around EGF (C), LIF and OSM (D), and VEGFA (E) loci superimposed on reads for Histone H3 lysine 4 mono methyl (H3K4me1), Histone H3 lysine 4 tri methyl (H3K4me3) and total H3 in myoblasts (pink) and myotubes (green).


F Snapshots of representative IGV tracks displaying RNA - Seq data from WT whole muscle ( WM ) , single myofibers ( SF ) and satellite cells ( SC ) showing the expression of EGF , EGFR and VEGFA . Ckm and Myf6 are used as positive controls for whole muscle and single fibers . PDGFRA , a marker of fibro / adipogenic progenitor cells , is used as a control to show the lack of niche cells in the single myofiber samples .

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F Snapshots of representative IGV tracks displaying RNA-Seq data from WT whole muscle (WM), single myofibers (SF) and satellite cells (SC) showing the expression of EGF, EGFR and VEGFA. Ckm and Myf6 are used as positive controls for whole muscle and single fibers. PDGFRA, a marker of fibro/adipogenic progenitor cells, is used as a control to show the lack of niche cells in the single myofiber samples.


G - H RNA - Sequencing Reads Per Million ( RPM ) for EGF ( G ) and EGFR ( H ) in satellite cells and single myofibers . RNA - Sequencing libraries were prepared from 1000 satellite cells freshly - isolated by FACS or from a single myofiber as described in the Material and Methods . ( n = 3 biological replicates per group ) bars = mean + / - SD , two - tailed t - test ) .

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G-H RNA-Sequencing Reads Per Million (RPM) for EGF (G) and EGFR (H) in satellite cells and single myofibers. RNA-Sequencing libraries were prepared from 1000 satellite cells freshly-isolated by FACS or from a single myofiber as described in the Material and Methods. (n=3 biological replicates per group) bars=mean +/- SD, two-tailed t-test).


I Activation of EGFR in MuSCs on EDL myofibers treated with EGF compared to untreated control . Myofibers were cultured for 48 hours and were treated with 40ng / ml of EGF for 10 minutes before fixing and staining with antibodies for PAX7 and phospho - EGFR . Scale bar = 30μm .

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I Activation of EGFR in MuSCs on EDL myofibers treated with EGF compared to untreated control. Myofibers were cultured for 48 hours and were treated with 40ng/ml of EGF for 10 minutes before fixing and staining with antibodies for PAX7 and phospho-EGFR. Scale bar=30μm.


J Activation of EGFR signaling in myoblasts treated with recombinant EGF . Myoblasts were treated with 2 ng / ml of recombinant protein in growth media ( Ham ' s F10 supplemented with 20 % FBS , 2 . 5 ng / ml bFGF ) for 15 minutes and lysed in RIPA lysis buffer . Western Blots were performed with antibodies against EGFR and phospho - EGFR ( pEGFR - Y1068 ) .

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J Activation of EGFR signaling in myoblasts treated with recombinant EGF. Myoblasts were treated with 2 ng/ml of recombinant protein in growth media (Ham's F10 supplemented with 20% FBS, 2.5 ng/ml bFGF) for 15 minutes and lysed in RIPA lysis buffer. Western Blots were performed with antibodies against EGFR and phospho-EGFR (pEGFR-Y1068).


K Depletion of Myf6 transcript by siRNAs in differentiated primary myotubes ( 5DM ) . ( n = 3 biological replicates ) , two - tailed t - test , bars = mean + / - SD L Depletion of Myf6 expression by siRNAs leads to a significant reduction in the gene expression output of Epidermal Growth Factor ( EGF ) . ( n = 3 biological replicates ) , two - tailed t - test , bars = mean + / -

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K Depletion of Myf6 transcript by siRNAs in differentiated primary myotubes (5DM). (n=3 biological replicates), two-tailed t-test, bars = mean +/- SD L Depletion of Myf6 expression by siRNAs leads to a significant reduction in the gene expression output of Epidermal Growth Factor (EGF). (n=3 biological replicates), two-tailed t-test, bars = mean +/- SD


B Picture of 8 week - old Myf6 - KO and WT mice .

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B Picture of 8 week-old Myf6-KO and WT mice.


C Relative expression of Myf6 transcript in the hindlimb skeletal muscles of Myf6 - KO and WT mice measured by Quantitative Real - Time PCR ( RT - qPCR ) ( n = 4 mice ) . Boxes represent 1st and 3rd quartile , central line represents the median , whiskers represent the range , two - tailed t - test .

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C Relative expression of Myf6 transcript in the hindlimb skeletal muscles of Myf6-KO and WT mice measured by Quantitative Real-Time PCR (RT-qPCR) (n=4 mice). Boxes represent 1st and 3rd quartile, central line represents the median, whiskers represent the range, two-tailed t-test.


D Color map of genes that are up / down regulated in the whole muscle transcriptome ( RNA - Seq ) of injured and uninjured hindlimb muscles from Myf6 - KO and WT counterparts . Differential expression is Log2 transformed and truncated at + / - 3 . Color scale goes from blue ( - 3 = lower than mean ) , to yellow ( + 3 = higher than mean ) . E Heat map showing the distribution of Myf6 peaks within 100kb of the TSS of the myokine genes ranging from zero ( black ) to six ChIP - Seq peaks ( red ) occupancy .

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D Color map of genes that are up/down regulated in the whole muscle transcriptome (RNA-Seq) of injured and uninjured hindlimb muscles from Myf6-KO and WT counterparts. Differential expression is Log2 transformed and truncated at +/-3. Color scale goes from blue (-3=lower than mean), to yellow (+3=higher than mean). E Heat map showing the distribution of Myf6 peaks within 100kb of the TSS of the myokine genes ranging from zero (black) to six ChIP-Seq peaks (red) occupancy.


F Expression of EGF protein in the blood serum from Myf6 - KO and heterozygous counterparts as measured by Enzyme - Linked Immunosorbent Assay ( ELISA ) . ( n = 5 heterozygous biological replicates , n = 3 Myf6 - KO biological replicates ) , two - tailed t - test , error bars = + / - SD G Expression of VEGFA protein in the blood serum from Myf6 - KO and heterozygous counterparts as measured by Enzyme - Linked Immunosorbent Assay ( ELISA ) . ( n = 5 heterozygous biological replicates , n = 3 Myf6 - KO biological replicates ) , two - tailed t - test , error bars = + / - SD .

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F Expression of EGF protein in the blood serum from Myf6-KO and heterozygous counterparts as measured by Enzyme-Linked Immunosorbent Assay (ELISA). (n=5 heterozygous biological replicates, n=3 Myf6-KO biological replicates), two-tailed t-test, error bars = +/- SD G Expression of VEGFA protein in the blood serum from Myf6-KO and heterozygous counterparts as measured by Enzyme-Linked Immunosorbent Assay (ELISA). (n=5 heterozygous biological replicates, n=3 Myf6-KO biological replicates), two-tailed t-test, error bars = +/- SD.


H Expression of VEGFA protein in the secretome of Myf6 - KO and WT myotubes as measured by enzyme - linked immunosorbent assay ( ELISA ) . Primary myotubes were derived from MuSCs isolated by Fluorescence Activated Cell Sorting ( FACS , see the Extended Material and Methods ) from Myf6 - KO and WT mice . Myotubes were obtained by feeding > 90 % confluent primary myoblasts with differentiation media ( DMEM supplemented with 5 % horse serum ) for seven days . Media from the last 48 hours of differentiation ( days 5 to 7 ) was collected for ELISA secretome analysis . Protein readouts were normalized to the total number of nuclei in each plate . ( n = 4 WT biological replicates , n = 3 Myf6 - KO biological replicates ) , two - tailed t - test , error bars = + / - SD I Expression of EGF protein in the secretome of Myf6 - KO and WT myotubes as measured by enzyme - linked immunosorbent assay ( ELISA ) . Primary myotubes were derived from MuSCs isolated by Fluorescence Activated Cell Sorting ( FACS , see the Extended Material and Methods ) from Myf6 - KO and WT mice . Myotubes were obtained by feeding > 90 % confluent primary myoblasts with differentiation media ( DMEM supplemented with 5 % horse serum ) for seven days . Media from the last 48 hours of differentiation ( days 5 to 7 ) was collected for ELISA secretome analysis . Protein readouts were normalized to the total number of nuclei in each plate . ( n = 4 WT biological replicates , n = 3 Myf6 - KO biological replicates ) , two - tailed t - test , error bars = + / - SD J Expression of EGF protein in the secretome of Myf6 - HF expressing myotubes derived from Myf6 - KO myoblasts ( rescue ) compared to Myf6 - KO myotubes as measured by enzyme - linked immunosorbent assay ( ELISA ) . Primary myotubes were derived from MuSCs isolated by Fluorescence Activated Cell Sorting ( FACS , see the Extended Material and Methods ) from Myf6 - KO mice . Myotubes were obtained by feeding > 90 % confluent primary myoblasts with differentiation media ( DMEM supplemented with 5 % horse serum ) for five days . Media from the last 48 hours of differentiation ( days 3 to 5 ) was collected for ELISA secretome analysis . Protein readouts were normalized to the total number of nuclei in each plate . ( n = 3 Myf6 - KO technical replicates , n = 4 rescue technical replicates ) , two - tailed t - test , error bars = + / - SD .

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

H Expression of VEGFA protein in the secretome of Myf6-KO and WT myotubes as measured by enzyme-linked immunosorbent assay (ELISA). Primary myotubes were derived from MuSCs isolated by Fluorescence Activated Cell Sorting (FACS, see the Extended Material and Methods) from Myf6-KO and WT mice. Myotubes were obtained by feeding >90% confluent primary myoblasts with differentiation media (DMEM supplemented with 5% horse serum) for seven days. Media from the last 48 hours of differentiation (days 5 to 7) was collected for ELISA secretome analysis. Protein readouts were normalized to the total number of nuclei in each plate. (n=4 WT biological replicates, n=3 Myf6-KO biological replicates), two-tailed t-test, error bars = +/- SD I Expression of EGF protein in the secretome of Myf6-KO and WT myotubes as measured by enzyme-linked immunosorbent assay (ELISA). Primary myotubes were derived from MuSCs isolated by Fluorescence Activated Cell Sorting (FACS, see the Extended Material and Methods) from Myf6-KO and WT mice. Myotubes were obtained by feeding >90% confluent primary myoblasts with differentiation media (DMEM supplemented with 5% horse serum) for seven days. Media from the last 48 hours of differentiation (days 5 to 7) was collected for ELISA secretome analysis. Protein readouts were normalized to the total number of nuclei in each plate. (n=4 WT biological replicates, n=3 Myf6-KO biological replicates), two-tailed t-test, error bars = +/- SD J Expression of EGF protein in the secretome of Myf6-HF expressing myotubes derived from Myf6-KO myoblasts (rescue) compared to Myf6-KO myotubes as measured by enzyme-linked immunosorbent assay (ELISA). Primary myotubes were derived from MuSCs isolated by Fluorescence Activated Cell Sorting (FACS, see the Extended Material and Methods) from Myf6-KO mice. Myotubes were obtained by feeding >90% confluent primary myoblasts with differentiation media (DMEM supplemented with 5% horse serum) for five days. Media from the last 48 hours of differentiation (days 3 to 5) was collected for ELISA secretome analysis. Protein readouts were normalized to the total number of nuclei in each plate. (n=3 Myf6-KO technical replicates, n=4 rescue technical replicates), two-tailed t-test, error bars = +/- SD.


K RT - qPCR of Myf6 expression in Myf6 - KO ( n = 2 technical replicates ) , WT ( n = 3 biological replicates ) and Myf6 - HF ( rescue ) myotubes ( n = 3 technical replicates ) ( 5DM ) . " Rescue " myotubes were generated from Myf6 - KO myoblasts infected with a retrovirus containing Myf6 - HF . two - tailed t - test , error bars = + / -

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K RT-qPCR of Myf6 expression in Myf6-KO (n=2 technical replicates), WT (n=3 biological replicates) and Myf6-HF (rescue) myotubes (n=3 technical replicates) (5DM). "Rescue" myotubes were generated from Myf6-KO myoblasts infected with a retrovirus containing Myf6-HF. two-tailed t-test, error bars = +/- SD


L - M RT - qPCR of EGF ( L ) and VEGFA ( M ) expression in Myf6 - KO ( n = 5 ) and rescue ( n = 3 - 4 ) primary myotubes ( 5DM ) . two - tailed t - test , error bars = + / -

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L-M RT-qPCR of EGF (L) and VEGFA (M) expression in Myf6-KO (n=5) and rescue (n=3-4) primary myotubes (5DM). two-tailed t-test, error bars = +/- SD


Immunofluorescence staining of WT ( N ) and Myf6 - KO EDL ( O ) myofibers stained with PAX7 , EGFR , and phospho - EGFR antibodies . Live fibers were maintained in culture media for 44 hours , followed by 4 hours of serum starving in non - supplemented DMEM . For treated samples , 40ng / ml of recombinant EGF was added to the non - supplemented DMEM for 10 minutes before fixation . Activation status of EGFR was assessed using antibodies against phospho - EGFR ( pEGFR - Y1068 ) . Scale bar = 30μm P Quantification of the percentage of all pEGFR + / PAX7 + cells ( defined as PAX7 + cells showing low or high pEGFR signal ) on WT ( n = 24 fibers from n = 3 mice ) , Myf6 - KO ( n = 22 fibers from n = 3 mice ) and Myf6 - KO EDL myofibers treated with EGF ( n = 14 fibers from n = 3 mice ) . two - tailed t - test , error bars = + / - SD Q Quantification of the percentage of EGFRhigh / PAX7 + cells ( defined as PAX7 + cells showing high pEGFR signal ) on WT ( n = 37 fibers from n = 3 mice ) , Myf6 - KO ( n = 34 fibers from n = 3 mice ) and Myf6 - KO EDL myofibers treated with EGF ( n = 11 fibers from n = 3 mice ) . two - tailed t - test , error bars = + / -

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Immunofluorescence staining of WT (N) and Myf6-KO EDL (O) myofibers stained with PAX7, EGFR, and phospho-EGFR antibodies. Live fibers were maintained in culture media for 44 hours, followed by 4 hours of serum starving in non-supplemented DMEM. For treated samples, 40ng/ml of recombinant EGF was added to the non-supplemented DMEM for 10 minutes before fixation. Activation status of EGFR was assessed using antibodies against phospho-EGFR (pEGFR-Y1068). Scale bar=30μm P Quantification of the percentage of all pEGFR+/PAX7+ cells (defined as PAX7+ cells showing low or high pEGFR signal) on WT (n=24 fibers from n=3 mice) , Myf6-KO (n=22 fibers from n=3 mice) and Myf6-KO EDL myofibers treated with EGF (n=14 fibers from n=3 mice). two-tailed t-test, error bars = +/- SD Q Quantification of the percentage of EGFRhigh/PAX7+ cells (defined as PAX7+ cells showing high pEGFR signal) on WT (n=37 fibers from n=3 mice), Myf6-KO (n=34 fibers from n=3 mice) and Myf6-KO EDL myofibers treated with EGF (n=11 fibers from n=3 mice). two-tailed t-test, error bars = +/- SD


A Immunofluorescent analysis of TA muscle cross - sections from Myf6 - KO and WT mice with PAX7 and Laminin ( LAMA1 ) antibodies at postnatal day 7 . Arrowheads indicate PAX7 + satellite cells . Scale bar = 200μm B Quantification of the number of PAX7 + cells per unit area ( mm2 ) in TA muscle cross - sections from Myf6 - KO and WT counterparts at postnatal day 7 . ( n = 30 fields of view from n = 3 animals ) , two - tailed t - test , error bars = + / - SD . C Immunofluorescent analysis of TA muscle cross - sections from Myf6 - KO and WT mice with PAX7 and Laminin ( LAMA1 ) antibodies at postnatal day 21 . Arrowheads indicate PAX7 + satellite cells . Scale bar = 200μm D Quantification of the number of PAX7 + cells per unit area ( mm2 ) in TA muscle cross - sections from Myf6 - KO and WT counterparts at postnatal day 21 . ( n = 27 fields of view from n = 3 animals ) , two tailed t - test , error bars = + / - SD ) . E Immunofluorescent analysis of TA muscle cross - sections from Myf6 - KO and WT mice with PAX7 and Laminin ( LAMA1 ) antibodies at 3 months of age . Arrowheads indicate PAX7 + satellite cells . Scale bar = 200μm F Quantification of the number of PAX7 + cells per unit area ( mm2 ) in TA muscle cross - sections from Myf6 - KO and WT counterparts at 3 months of age . ( n = 52 fields of view from n = 5 animals ) , two tailed t - test , error bars = + / - SD . G Immunofluorescent analysis of TA muscle cross - sections from Myf6 - KO and WT mice with PAX7 and Laminin ( LAMA1 ) antibodies at 10 months of age . Arrowheads indicate PAX7 + satellite cells . Scale bar = 200μm H Quantification of the number of PAX7 + cells per unit area ( mm2 ) in TA muscle cross - sections from Myf6 - KO and WT counterparts at 10 months of age . ( n = 60 fields of view from n = 3 animals ) , two tailed t - test , error bars = + / - SD .

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A Immunofluorescent analysis of TA muscle cross-sections from Myf6-KO and WT mice with PAX7 and Laminin (LAMA1) antibodies at postnatal day 7. Arrowheads indicate PAX7+ satellite cells. Scale bar=200μm B Quantification of the number of PAX7+ cells per unit area (mm2) in TA muscle cross-sections from Myf6-KO and WT counterparts at postnatal day 7. (n=30 fields of view from n=3 animals), two-tailed t-test, error bars = +/- SD. C Immunofluorescent analysis of TA muscle cross-sections from Myf6-KO and WT mice with PAX7 and Laminin (LAMA1) antibodies at postnatal day 21. Arrowheads indicate PAX7+ satellite cells. Scale bar=200μm D Quantification of the number of PAX7+ cells per unit area (mm2) in TA muscle cross-sections from Myf6-KO and WT counterparts at postnatal day 21. (n=27 fields of view from n=3 animals) , two tailed t-test, error bars = +/- SD). E Immunofluorescent analysis of TA muscle cross-sections from Myf6-KO and WT mice with PAX7 and Laminin (LAMA1) antibodies at 3 months of age. Arrowheads indicate PAX7+ satellite cells. Scale bar=200μm F Quantification of the number of PAX7+ cells per unit area (mm2) in TA muscle cross-sections from Myf6-KO and WT counterparts at 3 months of age. (n=52 fields of view from n=5 animals), two tailed t-test, error bars = +/- SD. G Immunofluorescent analysis of TA muscle cross-sections from Myf6-KO and WT mice with PAX7 and Laminin (LAMA1) antibodies at 10 months of age. Arrowheads indicate PAX7+ satellite cells. Scale bar=200μm H Quantification of the number of PAX7+ cells per unit area (mm2) in TA muscle cross-sections from Myf6-KO and WT counterparts at 10 months of age. (n=60 fields of view from n=3 animals), two tailed t-test, error bars = +/- SD.


Immunofluorescent analysis of myofiber - associated MuSCs isolated from the EDL muscles of 3 month - old Myf6 - KO and WT counterparts . Fibers were stained with PAX7 antibody and counterstained with DAPI at time T = 0 hours post fiber isolation . Scale bar = 25μm J Quantification of the number of MuSCs per fiber between 3 month - old Myf6 - KO ( n = 41 fibers from n = 3 mice ) and WT ( n = 31 fibers from n = 3 mice ) counterparts . Two - tailed t - test , error bars = + / - SD K Immunofluorescent analysis of myofiber - associated MuSCs isolated from the EDL muscles of 10 month - old Myf6 - KO and WT counterparts . Fibers were stained with PAX7 antibody and counterstained with DAPI at time T = 0 hours post fiber isolation . Scale bar = 25μm L Quantification of the number of MuSCs per fiber between 10 month - old Myf6 - KO ( n = 38 fibers from n = 2 mice ) and WT ( n = 67 fibers from n = 3 mice ) counterparts . Two - tailed t - test , error bars = + / -

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Immunofluorescent analysis of myofiber-associated MuSCs isolated from the EDL muscles of 3 month-old Myf6-KO and WT counterparts. Fibers were stained with PAX7 antibody and counterstained with DAPI at time T=0 hours post fiber isolation. Scale bar=25μm J Quantification of the number of MuSCs per fiber between 3 month-old Myf6-KO (n=41 fibers from n=3 mice) and WT (n=31 fibers from n=3 mice) counterparts. Two-tailed t-test, error bars = +/- SD K Immunofluorescent analysis of myofiber-associated MuSCs isolated from the EDL muscles of 10 month-old Myf6-KO and WT counterparts. Fibers were stained with PAX7 antibody and counterstained with DAPI at time T=0 hours post fiber isolation. Scale bar=25μm L Quantification of the number of MuSCs per fiber between 10 month-old Myf6-KO (n=38 fibers from n=2 mice) and WT (n=67 fibers from n=3 mice) counterparts. Two-tailed t-test, error bars = +/- SD


Immunofluorescent analysis of TA muscle cross - sections from Myf6 - KO and WT mice with PAX7 and KI67 antibodies at postnatal day 7 . Scale bar = 100μm , arrowheads highlight select MuSC B Quantification of the percentage of KI67 + over PAX7 + MuSCs between Myf6 - KO and WT mice at postnatal day 7 ( n = 15 fields of view from n = 3 animals ) Two - tailed t - test , error bars = + / - SD . C Immunofluorescent analysis of TA muscle cross - sections from Myf6 - KO and WT mice with PAX7 and KI67 antibodies at postnatal day 21 . Scale bar = 100μm , arrowheads highlight select MuSC D Quantification of the percentage of KI67 + over PAX7 + MuSCs between Myf6 - KO ( n = 28 fields of view from n = 3 animals ) and WT mice ( n = 31 fields of view from n = 3 animals ) at postnatal day 21 . Two - tailed t - test , error bars = + / - SD E Immunofluorescent analysis of TA muscle cross - sections from Myf6 - KO and WT mice with PAX7 and KI67 antibodies at 3 months of age . Scale bar = 100μm , arrowheads highlight select MuSC F Quantification of the percentage of KI67 + over PAX7 + MuSCs between Myf6 - KO and WT mice at 3 months of age ( n = 30 fields of view from n = 5 mice ) . Two - tailed t - test , error bars = + / -

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Immunofluorescent analysis of TA muscle cross-sections from Myf6-KO and WT mice with PAX7 and KI67 antibodies at postnatal day 7. Scale bar=100μm, arrowheads highlight select MuSC B Quantification of the percentage of KI67+ over PAX7+ MuSCs between Myf6-KO and WT mice at postnatal day 7 (n=15 fields of view from n=3 animals) Two-tailed t-test, error bars = +/- SD. C Immunofluorescent analysis of TA muscle cross-sections from Myf6-KO and WT mice with PAX7 and KI67 antibodies at postnatal day 21. Scale bar=100μm, arrowheads highlight select MuSC D Quantification of the percentage of KI67+ over PAX7+ MuSCs between Myf6-KO (n=28 fields of view from n=3 animals) and WT mice (n=31 fields of view from n=3 animals) at postnatal day 21. Two-tailed t-test, error bars = +/- SD E Immunofluorescent analysis of TA muscle cross-sections from Myf6-KO and WT mice with PAX7 and KI67 antibodies at 3 months of age. Scale bar=100μm, arrowheads highlight select MuSC F Quantification of the percentage of KI67+ over PAX7+ MuSCs between Myf6-KO and WT mice at 3 months of age (n=30 fields of view from n=5 mice). Two-tailed t-test, error bars = +/- SD


H ( Left ) Heat Map of up and down - regulated genes in the p38 MAPK pathway ( WP400 ) in Myf6 - KO and WT satellite cells . ( Right ) Gene set enrichment analysis of p38 MAPK pathway genes . In each plot , the blue curve represents the gene set enrichment score profile ( Subramanian et al . , 2005 ) across p38 MAPK pathway genes that are ranked by their differential expression . The grey curve represents the null expectation . P - values are based on permutation test ( 10 , 000 permutations ) .

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H (Left) Heat Map of up and down-regulated genes in the p38 MAPK pathway (WP400) in Myf6-KO and WT satellite cells. (Right) Gene set enrichment analysis of p38 MAPK pathway genes. In each plot, the blue curve represents the gene set enrichment score profile (Subramanian et al., 2005) across p38 MAPK pathway genes that are ranked by their differential expression. The grey curve represents the null expectation. P-values are based on permutation test (10,000 permutations).


Immunofluorescent analysis of WT and Myf6 - KO primary myotubes cultured in differentiation media for 7 days and stained with MF20 and DAPI . Scale bar = 200μm J Quantification of the fusion index between Myf6 - KO ( n = 54 fields of view from n = 3 mice ) and WT ( n = 81 fields of view from n = 4 mice ) 7DM myotubes . Fusion index is calculated as the percentage of myonuclei out of the total number of nuclei per field of view . Two - tailed t - test , error bars = + / -

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Immunofluorescent analysis of WT and Myf6-KO primary myotubes cultured in differentiation media for 7 days and stained with MF20 and DAPI. Scale bar=200μm J Quantification of the fusion index between Myf6-KO (n=54 fields of view from n=3 mice) and WT (n=81 fields of view from n=4 mice) 7DM myotubes. Fusion index is calculated as the percentage of myonuclei out of the total number of nuclei per field of view. Two-tailed t-test, error bars = +/- SD


EDL myofibers isolated from WT and Myf6 - KO mice fixed at time T = 0 and stained with PAX7 , MYOD and DAPI . Scale bar = 30μm L Quantification of the percentage of MYOD + / PAX7 + satellite cells per fiber in Myf6 - KO ( n = 88 myofibers ) vs WT ( n = 86 myofibers ) myofibers . Two - tailed t - test , error bars = + / - SD M Quantification of the average percentage of MYOD + / PAX7 + satellite cells per mouse in Myf6 - KO and WT myofibers ( n = 3 mice ) . Two - tailed t - test , error bars = + / -

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EDL myofibers isolated from WT and Myf6-KO mice fixed at time T=0 and stained with PAX7, MYOD and DAPI. Scale bar=30μm L Quantification of the percentage of MYOD+/PAX7+ satellite cells per fiber in Myf6-KO (n= 88 myofibers) vs WT (n= 86 myofibers) myofibers. Two-tailed t-test, error bars = +/- SD M Quantification of the average percentage of MYOD+/PAX7+ satellite cells per mouse in Myf6-KO and WT myofibers (n=3 mice). Two-tailed t-test, error bars = +/- SD


Staining for phospho - p38 MAPK on EDL myofibers cultured for 48 hours from Myf6 - KO and WT mice . Scale bar = 30μm O Quantification of the percentage of phospho - p38 + / PAX7 + satellite cells per fiber in Myf6 - KO ( n = 56 myofibers ) vs WT ( n = 67 myofibers ) myofibers . Two - tailed t - test , error bars = + / - SD P Quantification of the average percentage of phospho - p38 + / PAX7 + satellite cells per mouse in Myf6 - KO vs WT myofibers ( n = 3 mice ) . Two - tailed t - test , error bars = + / -

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Staining for phospho-p38 MAPK on EDL myofibers cultured for 48 hours from Myf6-KO and WT mice. Scale bar=30μm O Quantification of the percentage of phospho-p38+/PAX7+ satellite cells per fiber in Myf6-KO (n=56 myofibers) vs WT (n=67 myofibers) myofibers. Two-tailed t-test, error bars = +/- SD P Quantification of the average percentage of phospho-p38+/PAX7+ satellite cells per mouse in Myf6-KO vs WT myofibers (n=3 mice). Two-tailed t-test, error bars = +/- SD


Western blot analysis of C2C12 myoblasts treated with EGF , LIF , or EGF + LIF while in differentiation media and stained with phosphor - p38 , total p38 , myogenin , MF20 and GAPDH . Cells were treated with EGF ( 50ng / ml ) , LIF ( 1000U / ml ) or EGF + LIF after 48 hours in differentiation media , and every 6 hours following initial treatment , followed by cell lysis at 72 hours . R Phospho - p38 western blot signal intensity normalized to total p38 signal intensity in control , EGF - treated , LIF - treated and EGF + LIF - treated C2C12s . Signal intensity was measured by placing ROIs over western blot bands using LI - COR Image Studio . ( n = 3 technical replicates ) Two - tailed t - test , error bars = + / - SD S Myogenin western blot signal intensity normalized to GAPDH signal intensity in control , EGF - treated , LIF - treated and EGF + LIF - treated C2C12s . Signal intensity was measured by placing ROIs over western blot bands using LI - COR Image Studio . ( n = 3 technical replicates ) Two - tailed t - test , error bars = + / - SD T MF20 western blot signal intensity normalized to GAPDH signal intensity in control , EGF - treated , LIF - treated and EGF + LIF - treated C2C12s . Signal intensity was measured by placing ROIs over western blot bands using LI - COR Image Studio . ( n = 3 technical replicates ) Two - tailed t - test , error bars = + / -

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Western blot analysis of C2C12 myoblasts treated with EGF, LIF, or EGF+LIF while in differentiation media and stained with phosphor-p38, total p38, myogenin, MF20 and GAPDH. Cells were treated with EGF (50ng/ml), LIF (1000U/ml) or EGF+LIF after 48 hours in differentiation media, and every 6 hours following initial treatment, followed by cell lysis at 72 hours. R Phospho-p38 western blot signal intensity normalized to total p38 signal intensity in control, EGF-treated, LIF-treated and EGF+LIF-treated C2C12s. Signal intensity was measured by placing ROIs over western blot bands using LI-COR Image Studio. (n=3 technical replicates) Two-tailed t-test, error bars = +/- SD S Myogenin western blot signal intensity normalized to GAPDH signal intensity in control, EGF-treated, LIF-treated and EGF+LIF-treated C2C12s. Signal intensity was measured by placing ROIs over western blot bands using LI-COR Image Studio. (n=3 technical replicates) Two-tailed t-test, error bars = +/- SD T MF20 western blot signal intensity normalized to GAPDH signal intensity in control, EGF-treated, LIF-treated and EGF+LIF-treated C2C12s. Signal intensity was measured by placing ROIs over western blot bands using LI-COR Image Studio. (n=3 technical replicates) Two-tailed t-test, error bars = +/- SD


EDL myofibers treated repeatedly with recombinant EGF protein ( 100ng / ml ) at 0h , 24h , 32h , 40h , and 48h in culture , and fixed at 48 hours , compared to untreated controls . V Quantification of the number of PAX7 + cells per fiber in EGF - treated ( n = 19 fibers from n = 1 mouse ) and untreated control EDL myofibers ( n = 16 fibers from n = 1 mouse ) after 48 hours in culture . Two - tailed t - test , error bars = + / -

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EDL myofibers treated repeatedly with recombinant EGF protein (100ng/ml) at 0h, 24h, 32h, 40h, and 48h in culture, and fixed at 48 hours, compared to untreated controls. V Quantification of the number of PAX7+ cells per fiber in EGF-treated (n=19 fibers from n=1 mouse) and untreated control EDL myofibers (n=16 fibers from n=1 mouse) after 48 hours in culture. Two-tailed t-test, error bars = +/- SD


B Representative images of bioluminescent signal from hindlimbs of Myf6 - KO and WT counterparts at day 1 , 7 , 14 , and 21 post transplantation . Scale bar =

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B Representative images of bioluminescent signal from hindlimbs of Myf6-KO and WT counterparts at day 1, 7, 14, and 21 post transplantation. Scale bar=1cm


C Time course of average bioluminescent signal ( total flux in radiance ) in Myf6 - KO and WT mice at 1 , 7 , 14 and 21 days post MuSC transplantation showing engraftment dynamics of donor MuSCs ( mean across 4 - 5 animals per group ± SEM at each time point , two - tailed t - test ) .

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C Time course of average bioluminescent signal (total flux in radiance) in Myf6-KO and WT mice at 1, 7, 14 and 21 days post MuSC transplantation showing engraftment dynamics of donor MuSCs (mean across 4-5 animals per group ± SEM at each time point, two-tailed t-test).


D Bioluminescent signal ( radiance ) in Myf6 - KO and WT mice at each time point normalized to the bioluminescent signal at day 1 post MuSC transplantation . ( n = 4 - 5 animals per group ± SD ) two - tailed t - test , bars represent mean + / - SD .

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D Bioluminescent signal (radiance) in Myf6-KO and WT mice at each time point normalized to the bioluminescent signal at day 1 post MuSC transplantation. (n=4-5 animals per group ± SD) two-tailed t-test, bars represent mean +/- SD.


E - F Immunostaining of TA cross - sections of injured muscle from 3 month - old WT ( E ) and Myf6 - KO ( F ) mice stained with laminin ( LAMA1 ) and PAX7 antibodies . Acute muscle injury was performed by injection of Cardiotoxin ( CTX ) into the TA muscle of Myf6 - KO and WT mice ( n = 5 animals per group ) . Arrowheads indicate PAX7 + MuSCs . Scale bar = 100μm G Quantification of the number of PAX7 + cells per unit area of TA muscle cross sections between Myf6 - KO and WT mice ( n = 5 animals per group ) two - tailed t - test , bars represent mean + / -

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E-F Immunostaining of TA cross-sections of injured muscle from 3 month-old WT (E) and Myf6-KO (F) mice stained with laminin (LAMA1) and PAX7 antibodies. Acute muscle injury was performed by injection of Cardiotoxin (CTX) into the TA muscle of Myf6-KO and WT mice (n=5 animals per group). Arrowheads indicate PAX7+ MuSCs. Scale bar=100μm G Quantification of the number of PAX7+ cells per unit area of TA muscle cross sections between Myf6-KO and WT mice (n= 5 animals per group) two-tailed t-test, bars represent mean +/- SD


H Immunofluorescent analysis of Myf6 - KO and WT EDL myofibers cultured in growth media for 48 hours and stained with PAX7 and MYOD antibodies . Scale bar = 20μm IQuantification of the number of PAX7 + MuSCs per fiber in Myf6 - KO ( n = 27 fibers from n = 3 mice ) and WT ( n = 29 fibers from n = 3 mice ) mice after 48 hours in culture . Two - tailed t - test , error bars = + / - SD .

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H Immunofluorescent analysis of Myf6-KO and WT EDL myofibers cultured in growth media for 48 hours and stained with PAX7 and MYOD antibodies. Scale bar=20μm IQuantification of the number of PAX7+ MuSCs per fiber in Myf6-KO (n=27 fibers from n=3 mice) and WT (n=29 fibers from n=3 mice) mice after 48 hours in culture. Two-tailed t-test, error bars = +/- SD.


J Immunofluorescence staining of WT EDL myofibers cultured in growth media for 48 hours and treated with LIF ( 1000U / ml ) or EGF ( 80ng / ml ) every 8 hours . Fibers were stained with antibodies against PAX7 and MyoD . Scale bar = 30μm K Quantification of the number of PAX7 + and MYOD + cells on the EDL myofiber treated with recombinant LIF ( n = 80 fibers ) , EGF ( n = 50 fibers ) or vehicle control ( n = 61 fibers ) . Two - tailed t - test , bars represent mean + / -

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J Immunofluorescence staining of WT EDL myofibers cultured in growth media for 48 hours and treated with LIF (1000U/ml) or EGF (80ng/ml) every 8 hours. Fibers were stained with antibodies against PAX7 and MyoD. Scale bar=30μm K Quantification of the number of PAX7+ and MYOD+ cells on the EDL myofiber treated with recombinant LIF (n=80 fibers), EGF (n=50 fibers) or vehicle control (n=61 fibers). Two-tailed t-test, bars represent mean +/- SD


EdU staining of cultured primary myoblasts isolated by FACS ( see materials and methods ) from Myf6 - KO and WT mice . Staining was analyzed after 6 , 12 and 24 hours of EdU incorporation . Scale bar = 100μm M Quantification of the percentage of EdU + myoblasts after 6 hours ( WT n = 75 fields of view from n = 3 mice , Myf6 - KO n = 72 fields of view from n = 3 mice ) , 12 hours ( WT n = 82 fields of view from n = 3 mice , Myf6 - KOn = 82 fields of view from n = 3 mice ) , and 24 hours ( WT n = 70 fields of view from n = 3 mice , Myf6 - KO n = 65 fields of view from n = 3 mice ) of EdU incorporation . Scale bar =

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EdU staining of cultured primary myoblasts isolated by FACS (see materials and methods) from Myf6-KO and WT mice. Staining was analyzed after 6, 12 and 24 hours of EdU incorporation. Scale bar=100μm M Quantification of the percentage of EdU+ myoblasts after 6 hours (WT n=75 fields of view from n=3 mice, Myf6-KO n=72 fields of view from n=3 mice), 12 hours (WT n=82 fields of view from n=3 mice, Myf6-KOn=82 fields of view from n=3 mice), and 24 hours (WT n=70 fields of view from n=3 mice, Myf6-KO n=65 fields of view from n=3 mice) of EdU incorporation. Scale bar=100μm


Representative images of a co - culture assay between WT myotubes and either WT or Myf6 - KO myoblasts . Briefly , WT myoblasts were differentiated for 2 days before adding equal numbers of either WT or Myf6 - KO myoblasts for an additional 2 days , followed by immunostaining for MF20 and DAPI . Scale bar = 400μm O Quantification of the average number of myoblasts per mm2 that have remained mononuclear after their addition to 2DM WT myotubes , for 2 additional days ( n = 3 biological replicates ) . Two - tailed t - test , bars represent mean + / - SD .

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Representative images of a co-culture assay between WT myotubes and either WT or Myf6-KO myoblasts. Briefly, WT myoblasts were differentiated for 2 days before adding equal numbers of either WT or Myf6-KO myoblasts for an additional 2 days, followed by immunostaining for MF20 and DAPI. Scale bar=400μm O Quantification of the average number of myoblasts per mm2 that have remained mononuclear after their addition to 2DM WT myotubes, for 2 additional days (n=3 biological replicates). Two-tailed t-test, bars represent mean +/- SD.


( A , B ) Flag ‐ SENP3 or HA ‐ PELP1 was expressed in HeLa cells as indicated . Proteins were captured on Flag ‐ or HA ‐ beads and immunoblotted using antibodies directed against endogenous PELP1 or SENP3 as indicated .

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(A, B) Flag‐SENP3 or HA‐PELP1 was expressed in HeLa cells as indicated. Proteins were captured on Flag‐ or HA‐beads and immunoblotted using antibodies directed against endogenous PELP1 or SENP3 as indicated.


( C ) Endogenous PELP1 was immunoprecipitated from HeLa cells with a rabbit polyclonal antibody and immunocomplexes were probed for the presence of SENP3 by western blotting with an anti ‐ SENP3 antibody .

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(C) Endogenous PELP1 was immunoprecipitated from HeLa cells with a rabbit polyclonal antibody and immunocomplexes were probed for the presence of SENP3 by western blotting with an anti‐SENP3 antibody.


( D - F ) Flag ‐ or HA ‐ tagged proteins were expressed in HeLa cells , captured on Flag ‐ or HA ‐ beads and subjected to immunoblotting as indicated . In all experiments , the inputs represent 2 . 5 % of the total cell lysate .

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(D-F) Flag‐ or HA‐tagged proteins were expressed in HeLa cells, captured on Flag‐ or HA‐beads and subjected to immunoblotting as indicated. In all experiments, the inputs represent 2.5% of the total cell lysate.


( A , B ) Flag ‐ PELP1 and HA ‐ WDR18 were expressed in HeLa cells as indicated . HA ‐ WDR18 was captured on HA ‐ beads and the bound material was probed by immunoblotting for the presence of ectopically expressed Flag ‐ tagged PELP1 or endogenous PELP1 as indicated .

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(A, B) Flag‐PELP1 and HA‐WDR18 were expressed in HeLa cells as indicated. HA‐WDR18 was captured on HA‐beads and the bound material was probed by immunoblotting for the presence of ectopically expressed Flag‐tagged PELP1 or endogenous PELP1 as indicated.


( C ) Endogenous PELP1 was immunoprecipitated from HeLa cells with a rabbit polyclonal antibody and immunocomplexes were probed for the presence of endogenous WDR18 by western blotting with an anti ‐ WDR18 antibody .

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(C) Endogenous PELP1 was immunoprecipitated from HeLa cells with a rabbit polyclonal antibody and immunocomplexes were probed for the presence of endogenous WDR18 by western blotting with an anti‐WDR18 antibody.


( D ) Flag ‐ TEX10 was expressed with HA ‐ PELP1 in HeLa cells , captured on Flag ‐ beads and proteins were immunoblotted as indicated .

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(D) Flag‐TEX10 was expressed with HA‐PELP1 in HeLa cells, captured on Flag‐beads and proteins were immunoblotted as indicated.


( E ) HA ‐ TEX10 was expressed , captured on HA ‐ beads and associated proteins were probed for the presence of endogenous WDR18 using immunoblotting . The inputs represent 2 . 5 % of the total cell lysate .

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(E) HA‐TEX10 was expressed, captured on HA‐beads and associated proteins were probed for the presence of endogenous WDR18 using immunoblotting. The inputs represent 2.5% of the total cell lysate.


( A - C ) Immunofluorescence staining of endogenous PELP1 , SENP3 , UBF1 and NPM1 in HeLa cells . Nuclei were visualized using DAPI staining .

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(A-C) Immunofluorescence staining of endogenous PELP1, SENP3, UBF1 and NPM1 in HeLa cells. Nuclei were visualized using DAPI staining.


( B ) HeLa cells were transfected with siRNA duplexes targeting PELP1 , TEX10 , WDR18 , MDN1 or a control siRNA as indicated . Downregulation of the respective proteins was analysed by western blotting as indicated ( WB , three lower panels ) . At 72 h after transfection , cells were pulse labelled with 32P ‐ orthophosphate for 1 h and chased for 2 . 5 h . RNA was separated on a denaturing agarose gel and detected by ethidium bromide ( EtBr ) staining and autoradiography after drying of the gel .

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(B) HeLa cells were transfected with siRNA duplexes targeting PELP1, TEX10, WDR18, MDN1 or a control siRNA as indicated. Downregulation of the respective proteins was analysed by western blotting as indicated (WB, three lower panels). At 72 h after transfection, cells were pulse labelled with 32P‐orthophosphate for 1 h and chased for 2.5 h. RNA was separated on a denaturing agarose gel and detected by ethidium bromide (EtBr) staining and autoradiography after drying of the gel.


( C ) The signal intensities of the 28S and 32S rRNA forms were quantified by phosphoimager analysis and the ratio of 28S : 32S rRNA was calculated . Values represent the average of four ( two for siMDN1 ) independent experiments . Error bars indicate s . d .

(C) The signal intensities of the 28S and 32S rRNA forms were quantified by phosphoimager analysis and the ratio of 28S:32S rRNA was calculated. Values represent the average of four (two for siMDN1) independent experiments. Error bars indicate s.d.


( D ) PELP1 is associated with ITS2 ‐ containing RNA species . Endogenous PELP1 was immunoprecipitated from HeLa cells . In total , 10 % of the immunoprecipitated material was analysed by western blotting ( lower panel ) . The remaining material was used for RNA isolation and cDNA synthesis . The cDNA was used as template for PCR amplification using primers within the ITS2 / 28S region or GAPDH as a control . Input corresponds to 10 % of the cell lysate . Vertical line in the upper panel indicates removal of irrelevant adjacent lanes .

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(D) PELP1 is associated with ITS2‐containing RNA species. Endogenous PELP1 was immunoprecipitated from HeLa cells. In total, 10% of the immunoprecipitated material was analysed by western blotting (lower panel). The remaining material was used for RNA isolation and cDNA synthesis. The cDNA was used as template for PCR amplification using primers within the ITS2/28S region or GAPDH as a control. Input corresponds to 10% of the cell lysate. Vertical line in the upper panel indicates removal of irrelevant adjacent lanes.


( E ) Pre ‐ ribosomal particles were separated by sucrose gradient centrifugation of nuclear extracts . Proteins were recovered from the fractions by TCA precipitation and further analysed by SDS - PAGE and western blotting using antibodies as indicated . Vertical lines solely indicate loading onto two separate gels .

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(E) Pre‐ribosomal particles were separated by sucrose gradient centrifugation of nuclear extracts. Proteins were recovered from the fractions by TCA precipitation and further analysed by SDS-PAGE and western blotting using antibodies as indicated. Vertical lines solely indicate loading onto two separate gels.


( F ) Nucleolar export of pre ‐ 60S particles is strongly compromised in cells depleted of PELP1 , TEX10 , WDR18 , MDN1 or SENP3 . HeLa cells expressing YFP ‐ RpL27 from a tetracycline ‐ inducible promoter were transfected with the indicated siRNAs . After 24 h , expression of YFP ‐ RpL27 was induced and 72 h later the localization of RpL27 was monitored . Pictures were taken using identical exposure times .

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(F) Nucleolar export of pre‐60S particles is strongly compromised in cells depleted of PELP1, TEX10, WDR18, MDN1 or SENP3. HeLa cells expressing YFP‐RpL27 from a tetracycline‐inducible promoter were transfected with the indicated siRNAs. After 24 h, expression of YFP‐RpL27 was induced and 72 h later the localization of RpL27 was monitored. Pictures were taken using identical exposure times.


( A ) HA ‐ PELP1 was expressed alone or in combination with SUMO1 or SUMO2 in the presence or absence of SV5 ‐ tagged SENP3 in HeLa cells . Expression of the respective proteins was verified by western blotting . Detection of β ‐ tubulin served as loading control .

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(A) HA‐PELP1 was expressed alone or in combination with SUMO1 or SUMO2 in the presence or absence of SV5‐tagged SENP3 in HeLa cells. Expression of the respective proteins was verified by western blotting. Detection of β‐tubulin served as loading control.


( B ) SUMO modification of endogenous PELP1 was monitored by anti ‐ PELP1 immunoblotting in HeLa cells transfected with siRNAs as indicated . Depletion of the respective proteins was verified by western blotting .

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(B) SUMO modification of endogenous PELP1 was monitored by anti‐PELP1 immunoblotting in HeLa cells transfected with siRNAs as indicated. Depletion of the respective proteins was verified by western blotting.


( C ) HeLa cells were transfected with siRNAs and plasmids as indicated . His ‐ SUMO conjugates were recovered on Ni ‐ NTA beads and subjected to western blotting using anti ‐ PELP1 antibodies .

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(C) HeLa cells were transfected with siRNAs and plasmids as indicated. His‐SUMO conjugates were recovered on Ni‐NTA beads and subjected to western blotting using anti‐PELP1 antibodies.


( D ) HA ‐ PELP1 or HA ‐ PELP1K826R was expressed in the presence of SUMO1 or SUMO2 in HeLa cells . Expression of the respective proteins was analysed by western blotting . Detection of β ‐ tubulin served as loading control .

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(D) HA‐PELP1 or HA‐PELP1K826R was expressed in the presence of SUMO1 or SUMO2 in HeLa cells. Expression of the respective proteins was analysed by western blotting. Detection of β‐tubulin served as loading control.


( E , F ) Flag ‐ p14ARF and SUMO were expressed in HeLa cells and SUMO modification of ectopically expressed HA ‐ PELP1 or endogenous PELP1 was monitored by immunoblotting with anti ‐ HA or anti ‐ PELP1 antibodies as indicated . Arrowheads indicate SUMO - PELP1 conjugates .

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(E, F) Flag‐p14ARF and SUMO were expressed in HeLa cells and SUMO modification of ectopically expressed HA‐PELP1 or endogenous PELP1 was monitored by immunoblotting with anti‐HA or anti‐PELP1 antibodies as indicated. Arrowheads indicate SUMO-PELP1 conjugates.


( A ) Flag ‐ SUMO2 was expressed in HeLa cells by transient transfection . Flag ‐ SUMO2 ‐ expressing cells were visualized by indirect immunofluorescence . The localization of endogenous PELP1 was monitored in Flag ‐ SUMO2 ‐ positive cells ( green arrowheads ) or untransfected cells ( white arrowheads ) using anti ‐ PELP1 antibody .

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(A) Flag‐SUMO2 was expressed in HeLa cells by transient transfection. Flag‐SUMO2‐expressing cells were visualized by indirect immunofluorescence. The localization of endogenous PELP1 was monitored in Flag‐SUMO2‐positive cells (green arrowheads) or untransfected cells (white arrowheads) using anti‐PELP1 antibody.


( B - D ) HeLa cells were transfected with siRNAs as indicated . After fixation and permeabilization , the localization of PELP1 , LAS1L , TEX10 or PES1 was monitored by indirect immunofluorescence using the corresponding antibodies .

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(B-D) HeLa cells were transfected with siRNAs as indicated. After fixation and permeabilization, the localization of PELP1, LAS1L, TEX10 or PES1 was monitored by indirect immunofluorescence using the corresponding antibodies.


( E ) HeLa cells were depleted of endogenous PELP1 and reconstituted with siRNA resistant constructs expressing either wild ‐ type HA ‐ tagged PELP1 or a Flag ‐ tagged PELP1 - SUMO2 fusion protein . The localization of the respective proteins was monitored by indirect immunofluorescence using either anti ‐ HA ‐ or anti ‐ Flag ‐ antibodies .

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(E) HeLa cells were depleted of endogenous PELP1 and reconstituted with siRNA resistant constructs expressing either wild‐type HA‐tagged PELP1 or a Flag‐tagged PELP1-SUMO2 fusion protein. The localization of the respective proteins was monitored by indirect immunofluorescence using either anti‐HA‐ or anti‐Flag‐antibodies.


( F ) The localization of LAS1L or SENP3 was analysed in cells expressing Flag ‐ PELP1 - SUMO2 . Indirect immunofluorescence was done using anti ‐ LAS1L or anti ‐ SENP3 antibodies . Nuclei were counterstained with DAPI .

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(F) The localization of LAS1L or SENP3 was analysed in cells expressing Flag‐PELP1-SUMO2. Indirect immunofluorescence was done using anti‐LAS1L or anti‐SENP3 antibodies. Nuclei were counterstained with DAPI.


( A ) LAS1L was generated by in vitro transcription / translation and incubated with recombinant E1 and E2 enzymes and SUMO1 or SUMO2 , respectively , in the presence of ATP . In the control reaction ( lane 1 ) SUMO was not added .

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(A) LAS1L was generated by in vitro transcription/translation and incubated with recombinant E1 and E2 enzymes and SUMO1 or SUMO2, respectively, in the presence of ATP. In the control reaction (lane 1) SUMO was not added.


( B ) HeLa cells were transfected with siRNAs and plasmids as indicated . Expression of the respective proteins was verified by western blotting . Detection of β ‐ tubulin served as loading control .

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(B) HeLa cells were transfected with siRNAs and plasmids as indicated. Expression of the respective proteins was verified by western blotting. Detection of β‐tubulin served as loading control.


( C ) HeLa cells were transfected with siRNAs and plasmids as indicated . His ‐ SUMO2 conjugates were captured on magnetic Ni ‐ NTA beads and subjected to western blotting using anti ‐ LAS1L antibodies . Vertical lines indicate removal of irrelevant neighbouring lanes from the initial gel ( see Supplementary Figure 14 ) .

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(C) HeLa cells were transfected with siRNAs and plasmids as indicated. His‐SUMO2 conjugates were captured on magnetic Ni‐NTA beads and subjected to western blotting using anti‐LAS1L antibodies. Vertical lines indicate removal of irrelevant neighbouring lanes from the initial gel (see Supplementary Figure 14).


Electropherograms of the XRCC4 genomic region encompassing the nucleotide substitutions in available members of the family .

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Electropherograms of the XRCC4 genomic region encompassing the nucleotide substitutions in available members of the family.


Quantitative real - time PCR of XRCC4 relative to GAPDH mRNA in affected ( II - 1 and II - 2 ) and control ( CT1 and CT2 ) fibroblasts . Each value refers to the mean of three independent experiments performed in duplicate . Error bars indicate the standard deviation .

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Quantitative real-time PCR of XRCC4 relative to GAPDH mRNA in affected (II-1 and II-2) and control (CT1 and CT2) fibroblasts. Each value refers to the mean of three independent experiments performed in duplicate. Error bars indicate the standard deviation.


Immunoblot analysis of total lysates from fibroblasts ( fbs ) and of in vitro - synthesized proteins ( i . v . p ) . fl : in vitro - translated full - length XRCC4 protein ; ret . : reticulocyte lysate used for in vitro protein synthesis ; 225 * : in vitro - translated XRCC4 R225 * truncated protein ; Ct : lysate of control fbs ; and II - 1 and II - 2 : lysates of fbs from patient II - 1 and II - 2 . α - XRCC4 ( Abcam , 1 : 1 , 000 ) and α - GAPDH antibodies were used .

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Immunoblot analysis of total lysates from fibroblasts (fbs) and of in vitro-synthesized proteins (i.v.p). fl: in vitro-translated full-length XRCC4 protein; ret.: reticulocyte lysate used for in vitro protein synthesis; 225*: in vitro-translated XRCC4 R225* truncated protein; Ct: lysate of control fbs; and II-1 and II-2: lysates of fbs from patient II-1 and II-2. α-XRCC4 (Abcam, 1:1,000) and α-GAPDH antibodies were used.


Immunoblot analysis of total lysates from fibroblasts using α - LIG4 ( GeneTex , 1 : 1 , 000 ) and α - SDHA antibodies . Ct : lysate of control fbs ; II - 1 and II - 2 : lysates of fbs from patient II - 1 and II - 2 . The position of the molecular weight ( MW ) marker proteins is indicated .

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Immunoblot analysis of total lysates from fibroblasts using α-LIG4 (GeneTex, 1:1,000) and α-SDHA antibodies. Ct: lysate of control fbs; II-1 and II-2: lysates of fbs from patient II-1 and II-2. The position of the molecular weight (MW) marker proteins is indicated.


γ - H2AX nuclear foci in XRCC4wt and mutant fibroblasts ( XRCC4m / m1 and XRCC4m / m2 ) were determined at different times after irradiation with 0 . 5 Gy of γ - rays . Values , subtracted of their non - irradiated control ( 1 . 3 foci / nucleus in XRCC4wt cells and 1 . 4 and 0 . 9 foci / nucleus in XRCC4m / m1 and XRCC4m / m2 cells , respectively ) , are the mean ± SD of three independent experiments . Percentages of γ - H2AX foci in XRCC4wt and XRCC4m / m fibroblasts ( pooled values ) remaining at the indicated time - points . Immunofluorescence micrographs of γ - H2AX foci in XRCC4wt and XRCC4m / m fibroblasts . Data information : * * * P < 0 . 001 ; * * P < 0 . 01 ; * P < 0 . 05 ( XRCC4m / m vs . XRCC4wt cells ) , two - way ANOVA , Bonferroni post hoc test .

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γ-H2AX nuclear foci in XRCC4wt and mutant fibroblasts (XRCC4m/m1 and XRCC4m/m2) were determined at different times after irradiation with 0.5 Gy of γ-rays. Values, subtracted of their non-irradiated control (1.3 foci/nucleus in XRCC4wt cells and 1.4 and 0.9 foci/nucleus in XRCC4m/m1 and XRCC4m/m2 cells, respectively), are the mean ± SD of three independent experiments.Percentages of γ-H2AX foci in XRCC4wt and XRCC4m/m fibroblasts (pooled values) remaining at the indicated time-points.Immunofluorescence micrographs of γ-H2AX foci in XRCC4wt and XRCC4m/m fibroblasts.Data information: ***P < 0.001; **P < 0.01; *P < 0.05 (XRCC4m/m vs. XRCC4wt cells), two-way ANOVA, Bonferroni post hoc test.


A , B Percentages of γ - H2AX nuclear foci remaining in CENP - F - negative ( A ) and CENP - F - positive ( B ) cells at the indicated time - points after irradiation with 0 . 5 Gy of γ - rays . Values , subtracted of their non - irradiated controls , are the mean ± SD of three independent experiments . * * * P < 0 . 001 ; * P < 0 . 05 ( XRCC4m / m versus XRCC4wt cells ) , two - way ANOVA , Bonferroni post hoc test . C Immunofluorescence micrographs of γ - H2AX foci in CENP - F - positive and negative fibroblasts at 2 h after irradiation . Arrow heads indicate the CENP - F - positive cells .

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A, B Percentages of γ-H2AX nuclear foci remaining in CENP-F-negative (A) and CENP-F-positive (B) cells at the indicated time-points after irradiation with 0.5 Gy of γ-rays. Values, subtracted of their non-irradiated controls, are the mean ± SD of three independent experiments. ***P < 0.001; *P < 0.05 (XRCC4m/m versus XRCC4wt cells), two-way ANOVA, Bonferroni post hoc test.C Immunofluorescence micrographs of γ-H2AX foci in CENP-F-positive and negative fibroblasts at 2 h after irradiation. Arrow heads indicate the CENP-F-positive cells.


RAD51 foci in XRCC4wt and XRCC4m / m fibroblasts irradiated with 0 . 5 Gy of γ - rays . Values are the mean ± SD of three independent experiments . * * * P < 0 . 001 ; * * P < 0 . 01 ( XRCC4m / m versus XRCC4wt cells ) , two - way ANOVA , Bonferroni post hoc test . Immunofluorescence micrographs of RAD51 foci in XRCC4wt and XRCC4m / mfibroblasts . Arrowheads indicate the CENP - F - positive cells .

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RAD51 foci in XRCC4wt and XRCC4m/m fibroblasts irradiated with 0.5 Gy of γ-rays. Values are the mean ± SD of three independent experiments. ***P < 0.001; **P < 0.01 (XRCC4m/m versus XRCC4wt cells), two-way ANOVA, Bonferroni post hoc test.Immunofluorescence micrographs of RAD51 foci in XRCC4wt and XRCC4m/mfibroblasts. Arrowheads indicate the CENP-F-positive cells.


XRCC4wt and XRCC4m / m cells irradiated with 0 . 5 Gy of γ - rays were exposed to the PARP1 inhibitor 3 ′ - AB or to solvent ( DMSO ) . The γ - H2AX foci number is the mean ± SD of three independent experiments . * P < 0 . 05 ( XRCC4m / m + 3 ′ - AB versus XRCC4m / m + DMSO ) , * * * P < 0 . 001 ( XRCC4m / m + 3 ′ - AB versus XRCC4wt + 3 ′ - AB ) , two - way ANOVA , Bonferroni post hoc test .

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XRCC4wt and XRCC4m/m cells irradiated with 0.5 Gy of γ-rays were exposed to the PARP1 inhibitor 3′-AB or to solvent (DMSO). The γ-H2AX foci number is the mean ± SD of three independent experiments. *P < 0.05 (XRCC4m/m + 3′-AB versus XRCC4m/m + DMSO), ***P < 0.001 (XRCC4m/m + 3′-AB versus XRCC4wt + 3′-AB), two-way ANOVA, Bonferroni post hoc test.


A , B Percentages of DSBs repaired in increasing time intervals ( 0 . 5 - 2 h , 0 . 5 - 6 h , 0 . 5 - 24 h ) were calculated in XRCC4wt ( A ) and XRCC4m / m ( B ) cells from the number of γ - H2AX foci in presence or absence of a PARP - 1 inhibitor , 3 ′ - AB . * * P < 0 . 01 ( XRCC4m / m + 3 ′ - AB versus XRCC4m / m + DMSO ) , two - way ANOVA , Bonferroni post hoc test .

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A, B Percentages of DSBs repaired in increasing time intervals (0.5-2 h, 0.5-6 h, 0.5-24 h) were calculated in XRCC4wt (A) and XRCC4m/m (B) cells from the number of γ-H2AX foci in presence or absence of a PARP-1 inhibitor, 3′-AB. **P < 0.01 (XRCC4m/m + 3′-AB versus XRCC4m/m + DMSO), two-way ANOVA, Bonferroni post hoc test.


B Western blot analysis confirming two Mettl5 KO clones by specific METTL5 antibody . α - TUBULIN was used as a loading control .

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B Western blot analysis confirming two Mettl5 KO clones by specific METTL5 antibody. α-TUBULIN was used as a loading control.


C LC - MS / MS analysis of m6A / A levels in purified 18S rRNA and 28S rRNA from the WT and Mettl5 KO ( KO - 6B ) mESCs .

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C LC-MS/MS analysis of m6A/A levels in purified 18S rRNA and 28S rRNA from the WT and Mettl5 KO (KO-6B) mESCs.


D PAGE gel electrophoresis showing the PCR amplification of the elongated and ligated products of SELECT method for detecting m6A1832 site and A1825 site ( for input control ) in 18S rRNA of WT and Mettl5 KO ( KO - 6B ) mESCs , respectively .

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D PAGE gel electrophoresis showing the PCR amplification of the elongated and ligated products of SELECT method for detecting m6A1832 site and A1825 site (for input control) in 18S rRNA of WT and Mettl5 KO (KO-6B) mESCs, respectively.


E LC - MS / MS analysis of d3 - m6A / A levels in 18S rRNA oligos after being incubated with recombinant WT or catalytically mutant mouse METTL5 proteins in vitro . No enzyme group was used as a negative control .

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E LC-MS/MS analysis of d3-m6A/A levels in 18S rRNA oligos after being incubated with recombinant WT or catalytically mutant mouse METTL5 proteins in vitro. No enzyme group was used as a negative control.


A , B Representative bright - filed ( A ) and AP staining ( B ) images of the WT and Mettl5 KO mESCs cultured in 2i / Lif medium . Scale bars represent 100 μm and 50 μm , respectively .

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A,B Representative bright-filed (A) and AP staining (B) images of the WT and Mettl5 KO mESCs cultured in 2i/Lif medium. Scale bars represent 100 μm and 50 μm, respectively.


C RT - qPCR analysis of the expression of pluripotency genes in WT and Mettl5 KO mESCs . Data are represented as mean ± SD from three biological replicates . Student ' s t test , two - tailed . ns , not significant ; * * * , P < 0 . 001 .

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C RT-qPCR analysis of the expression of pluripotency genes in WT and Mettl5 KO mESCs. Data are represented as mean ± SD from three biological replicates. Student's t test, two-tailed. ns, not significant; ***, P<0.001.


D Western blot analysis of the expression of pluripotency markers in WT and Mettl5 KO mESCs . α - TUBULIN was used as a loading control .

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D Western blot analysis of the expression of pluripotency markers in WT and Mettl5 KO mESCs. α-TUBULIN was used as a loading control.


E Immunostaining analysis of the expression of pluripotency markers ( POU5F1 , SOX2 and NANOG ) in WT and Mettl5 KO mESCs , respectively . DAPI ( blue ) was used as a nuclear counterstain . Scale bars represent 10 μm .

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E Immunostaining analysis of the expression of pluripotency markers (POU5F1, SOX2 and NANOG) in WT and Mettl5 KO mESCs, respectively. DAPI (blue) was used as a nuclear counterstain. Scale bars represent 10 μm.


A Western blot analysis showing dynamic expression patterns of POU5F1 and METTL5 during the indicated time points of EB induction . α - TUBULIN was used as a loading control .

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A Western blot analysis showing dynamic expression patterns of POU5F1 and METTL5 during the indicated time points of EB induction. α-TUBULIN was used as a loading control.


B Histogram showing the threshold cycles of qPCR ( qPCRCT ) for detecting m6A1832 site in 18S rRNA during the indicated time points of EB induction . Data are shown as mean ± SD from three biological replicates . Student ' s t test , two - tailed . ns , not significant ; * , P < 0 . 05 .

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B Histogram showing the threshold cycles of qPCR (qPCRCT) for detecting m6A1832 site in 18S rRNA during the indicated time points of EB induction. Data are shown as mean ± SD from three biological replicates. Student's t test, two-tailed. ns, not significant; *, P<0.05.