I Activation of EGFR in MuSCs on EDL myofibers treated with EGF compared to untreated control . Myofibers were cultured for 48 hours were treated with 40ng / ml of EGF for 10 minutes before fixing with antibodies for PAX7 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 in growth media ( Ham ' s F10 supplemented with 20 % FBS , 2 . 5 ng / ml bFGF ) for 15 minutes lysed in RIPA lysis buffer . Western Blots were performed with antibodies against EGFR 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 myotubes ( 5DM ) . ( n = 3 biological replicates ) , two - t - , bars = mean + / - SD L Depletion of Myf6 expression by siRNAs leads to a significant reduction in gene expression output of Epidermal Growth Factor ( EGF ) . ( n = 3 biological replicates ) , two - t - , 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 WT mice .

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


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

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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 whole muscle transcriptome ( - Seq ) of injured uninjured hindlimb from Myf6 - KO WT counterparts . Differential expression is Log2 transformed truncated at + / - 3 . Color scale goes from blue ( - 3 = lower than mean ) , to yellow ( + 3 = higher than mean ) . E Heat map showing distribution of Myf6 peaks within 100kb of TSS of 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 in blood serum from Myf6 - KO heterozygous counterparts as measured by Enzyme - Linked Immunosorbent ( ELISA ) . ( n = 5 heterozygous biological replicates , n = 3 Myf6 - KO biological replicates ) , two - t - , error bars = + / - SD G Expression of VEGFA in blood serum from Myf6 - KO heterozygous counterparts as measured by Enzyme - Linked Immunosorbent ( ELISA ) . ( n = 5 heterozygous biological replicates , n = 3 Myf6 - KO biological replicates ) , two - t - , 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 in secretome of Myf6 - KO WT myotubes as measured by enzyme - linked immunosorbent ( ELISA ) . myotubes were derived from MuSCs isolated by Activated Sorting ( FACS , see Extended Material Methods ) from Myf6 - KO WT mice . Myotubes were obtained by feeding > 90 % confluent myoblasts with differentiation media ( DMEM supplemented with 5 % horse serum ) for seven days . Media from last 48 hours of differentiation ( days 5 to 7 ) was collected for ELISA secretome . readouts were normalized to total number of nuclei in each plate . ( n = 4 WT biological replicates , n = 3 Myf6 - KO biological replicates ) , two - t - , error bars = + / - SD I Expression of EGF in secretome of Myf6 - KO WT myotubes as measured by enzyme - linked immunosorbent ( ELISA ) . myotubes were derived from MuSCs isolated by Activated Sorting ( FACS , see Extended Material Methods ) from Myf6 - KO WT mice . Myotubes were obtained by feeding > 90 % confluent myoblasts with differentiation media ( DMEM supplemented with 5 % horse serum ) for seven days . Media from last 48 hours of differentiation ( days 5 to 7 ) was collected for ELISA secretome . readouts were normalized to total number of nuclei in each plate . ( n = 4 WT biological replicates , n = 3 Myf6 - KO biological replicates ) , two - t - , error bars = + / - SD J Expression of EGF in secretome of Myf6 - HF expressing myotubes derived from Myf6 - KO myoblasts ( rescue ) compared to Myf6 - KO myotubes as measured by enzyme - linked immunosorbent ( ELISA ) . myotubes were derived from MuSCs isolated by Activated Sorting ( FACS , see Extended Material Methods ) from Myf6 - KO mice . Myotubes were obtained by feeding > 90 % confluent myoblasts with differentiation media ( DMEM supplemented with 5 % horse serum ) for five days . Media from last 48 hours of differentiation ( days 3 to 5 ) was collected for ELISA secretome . readouts were normalized to total number of nuclei in each plate . ( n = 3 Myf6 - KO technical replicates , n = 4 rescue technical replicates ) , two - t - , error bars = + / - SD .

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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 ) 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 - t - , 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 ) VEGFA ( M ) expression in Myf6 - KO ( n = 5 ) rescue ( n = 3 - 4 ) myotubes ( 5DM ) . two - t - , 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 of WT ( N ) Myf6 - KO EDL ( O ) myofibers stained with PAX7 , EGFR , phospho - EGFR antibodies . Live 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 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 of of pEGFR + / PAX7 + ( defined as PAX7 + showing low or high pEGFR signal ) on WT ( n = 24 from n = 3 mice ) , Myf6 - KO ( n = 22 from n = 3 mice ) Myf6 - KO EDL myofibers treated with EGF ( n = 14 from n = 3 mice ) . two - t - , error bars = + / - SD Q of of EGFRhigh / PAX7 + ( defined as PAX7 + showing high pEGFR signal ) on WT ( n = 37 from n = 3 mice ) , Myf6 - KO ( n = 34 from n = 3 mice ) Myf6 - KO EDL myofibers treated with EGF ( n = 11 from n = 3 mice ) . two - t - , 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 of TA muscle cross - sections from Myf6 - KO WT mice with PAX7 Laminin ( LAMA1 ) antibodies at postnatal day 7 . Arrowheads indicate PAX7 + satellite . Scale bar = 200μm B of number of PAX7 + per unit ( mm2 ) in TA muscle cross - sections from Myf6 - KO WT counterparts at postnatal day 7 . ( n = 30 fields of view from n = 3 ) , two - t - , error bars = + / - SD . C Immunofluorescent of TA muscle cross - sections from Myf6 - KO WT mice with PAX7 Laminin ( LAMA1 ) antibodies at postnatal day 21 . Arrowheads indicate PAX7 + satellite . Scale bar = 200μm D of number of PAX7 + per unit ( mm2 ) in TA muscle cross - sections from Myf6 - KO WT counterparts at postnatal day 21 . ( n = 27 fields of view from n = 3 ) , two t - , error bars = + / - SD ) . E Immunofluorescent of TA muscle cross - sections from Myf6 - KO WT mice with PAX7 Laminin ( LAMA1 ) antibodies at 3 months of . Arrowheads indicate PAX7 + satellite . Scale bar = 200μm F of number of PAX7 + per unit ( mm2 ) in TA muscle cross - sections from Myf6 - KO WT counterparts at 3 months of . ( n = 52 fields of view from n = 5 ) , two t - , error bars = + / - SD . G Immunofluorescent of TA muscle cross - sections from Myf6 - KO WT mice with PAX7 Laminin ( LAMA1 ) antibodies at 10 months of . Arrowheads indicate PAX7 + satellite . Scale bar = 200μm H of number of PAX7 + per unit ( mm2 ) in TA muscle cross - sections from Myf6 - KO WT counterparts at 10 months of . ( n = 60 fields of view from n = 3 ) , two t - , 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 of myofiber - associated MuSCs isolated from EDL of 3 month - old Myf6 - KO WT counterparts . were stained with PAX7 antibody counterstained with DAPI at time T = 0 hours post fiber isolation . Scale bar = 25μm J of number of MuSCs per fiber between 3 month - old Myf6 - KO ( n = 41 from n = 3 mice ) WT ( n = 31 from n = 3 mice ) counterparts . Two - t - , error bars = + / - SD K Immunofluorescent of myofiber - associated MuSCs isolated from EDL of 10 month - old Myf6 - KO WT counterparts . were stained with PAX7 antibody counterstained with DAPI at time T = 0 hours post fiber isolation . Scale bar = 25μm L of number of MuSCs per fiber between 10 month - old Myf6 - KO ( n = 38 from n = 2 mice ) WT ( n = 67 from n = 3 mice ) counterparts . Two - t - , 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 of TA muscle cross - sections from Myf6 - KO WT mice with PAX7 KI67 antibodies at postnatal day 7 . Scale bar = 100μm , arrowheads highlight select MuSC B of of KI67 + over PAX7 + MuSCs between Myf6 - KO WT mice at postnatal day 7 ( n = 15 fields of view from n = 3 ) Two - t - , error bars = + / - SD . C Immunofluorescent of TA muscle cross - sections from Myf6 - KO WT mice with PAX7 KI67 antibodies at postnatal day 21 . Scale bar = 100μm , arrowheads highlight select MuSC D of of KI67 + over PAX7 + MuSCs between Myf6 - KO ( n = 28 fields of view from n = 3 ) WT mice ( n = 31 fields of view from n = 3 ) at postnatal day 21 . Two - t - , error bars = + / - SD E Immunofluorescent of TA muscle cross - sections from Myf6 - KO WT mice with PAX7 KI67 antibodies at 3 months of . Scale bar = 100μm , arrowheads highlight select MuSC F of of KI67 + over PAX7 + MuSCs between Myf6 - KO WT mice at 3 months of ( n = 30 fields of view from n = 5 mice ) . Two - t - , 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 down - regulated genes in p38 MAPK pathway ( WP400 ) in Myf6 - KO WT satellite . ( Right ) Gene set enrichment of p38 MAPK pathway genes . In each plot , blue curve represents gene set enrichment score profile ( Subramanian et al . , 2005 ) across p38 MAPK pathway genes that are ranked by their differential expression . grey curve represents null expectation . P - values are on permutation ( 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 of WT Myf6 - KO myotubes cultured in differentiation media for 7 days stained with MF20 DAPI . Scale bar = 200μm J of fusion index between Myf6 - KO ( n = 54 fields of view from n = 3 mice ) WT ( n = 81 fields of view from n = 4 mice ) 7DM myotubes . Fusion index is calculated as of myonuclei out of total number of nuclei per field of view . Two - t - , 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 Myf6 - KO mice fixed at time T = 0 stained with PAX7 , MYOD DAPI . Scale bar = 30μm L of of MYOD + / PAX7 + satellite per fiber in Myf6 - KO ( n = 88 myofibers ) vs WT ( n = 86 myofibers ) myofibers . Two - t - , error bars = + / - SD M of average of MYOD + / PAX7 + satellite per mouse in Myf6 - KO WT myofibers ( n = 3 mice ) . Two - t - , 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


for phospho - p38 MAPK on EDL myofibers cultured for 48 hours from Myf6 - KO WT mice . Scale bar = 30μm O of of phospho - p38 + / PAX7 + satellite per fiber in Myf6 - KO ( n = 56 myofibers ) vs WT ( n = 67 myofibers ) myofibers . Two - t - , error bars = + / - SD P of average of phospho - p38 + / PAX7 + satellite per mouse in Myf6 - KO vs WT myofibers ( n = 3 mice ) . Two - t - , 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 of C2C12 myoblasts treated with EGF , LIF , or EGF + LIF while in differentiation media stained with phosphor - p38 , total p38 , myogenin , MF20 GAPDH . were treated with EGF ( 50ng / ml ) , LIF ( 1000U / ml ) or EGF + LIF after 48 hours in differentiation media , every 6 hours following initial treatment , followed by lysis at 72 hours . R Phospho - p38 western blot signal normalized to total p38 signal in control , EGF - treated , LIF - treated EGF + LIF - treated C2C12s . Signal was measured by placing ROIs over western blot bands using LI - COR Image Studio . ( n = 3 technical replicates ) Two - t - , error bars = + / - SD S Myogenin western blot signal normalized to GAPDH signal in control , EGF - treated , LIF - treated EGF + LIF - treated C2C12s . Signal was measured by placing ROIs over western blot bands using LI - COR Image Studio . ( n = 3 technical replicates ) Two - t - , error bars = + / - SD T MF20 western blot signal normalized to GAPDH signal in control , EGF - treated , LIF - treated EGF + LIF - treated C2C12s . Signal was measured by placing ROIs over western blot bands using LI - COR Image Studio . ( n = 3 technical replicates ) Two - t - , 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 ( 100ng / ml ) at 0h , 24h , 32h , 40h , 48h in culture , fixed at 48 hours , compared to untreated controls . V of number of PAX7 + per fiber in EGF - treated ( n = 19 from n = 1 mouse ) untreated control EDL myofibers ( n = 16 from n = 1 mouse ) after 48 hours in culture . Two - t - , 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 of bioluminescent signal from hindlimbs of Myf6 - KO WT counterparts at day 1 , 7 , 14 , 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 WT mice at 1 , 7 , 14 21 days post MuSC transplantation showing engraftment dynamics of donor MuSCs ( mean across 4 - 5 per group ± at each time point , two - t - ) .

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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 WT mice at each time point normalized to bioluminescent signal at day 1 post MuSC transplantation . ( n = 4 - 5 per group ± SD ) two - t - , 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 ) Myf6 - KO ( F ) mice stained with laminin ( LAMA1 ) PAX7 antibodies . Acute muscle injury was performed by injection of Cardiotoxin ( CTX ) into TA muscle of Myf6 - KO WT mice ( n = 5 per group ) . Arrowheads indicate PAX7 + MuSCs . Scale bar = 100μm G of number of PAX7 + per unit of TA muscle cross sections between Myf6 - KO WT mice ( n = 5 per group ) two - t - , 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 of Myf6 - KO WT EDL myofibers cultured in growth media for 48 hours stained with PAX7 MYOD antibodies . Scale bar = 20μm IQuantification of number of PAX7 + MuSCs per fiber in Myf6 - KO ( n = 27 from n = 3 mice ) WT ( n = 29 from n = 3 mice ) mice after 48 hours in culture . Two - t - , 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 of WT EDL myofibers cultured in growth media for 48 hours treated with LIF ( 1000U / ml ) or EGF ( 80ng / ml ) every 8 hours . were stained with antibodies against PAX7 MyoD . Scale bar = 30μm K of number of PAX7 + MYOD + on EDL myofiber treated with recombinant LIF ( n = 80 ) , EGF ( n = 50 ) or vehicle control ( n = 61 ) . Two - t - , 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 of cultured myoblasts isolated by FACS ( see materials methods ) from Myf6 - KO WT mice . was analyzed after 6 , 12 24 hours of EdU incorporation . Scale bar = 100μm M of 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 ) , 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 of a co - culture between WT myotubes 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 DAPI . Scale bar = 400μm O of 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 - t - , 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 as indicated . were captured on Flag ‐ or HA ‐ beads 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 with a rabbit polyclonal antibody immunocomplexes were probed for 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 were expressed in HeLa , captured on Flag ‐ or HA ‐ beads subjected to immunoblotting as indicated . In experiments , inputs represent 2 . 5 % of total 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 HA ‐ WDR18 were expressed in HeLa as indicated . HA ‐ WDR18 was captured on HA ‐ beads bound material was probed by immunoblotting for 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 with a rabbit polyclonal antibody immunocomplexes were probed for 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 , captured on Flag ‐ beads 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 associated were probed for presence of endogenous WDR18 using immunoblotting . inputs represent 2 . 5 % of total 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 of endogenous PELP1 , SENP3 , UBF1 NPM1 in HeLa . Nuclei were visualized using DAPI .

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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 were transfected with siRNA duplexes targeting PELP1 , TEX10 , WDR18 , MDN1 or a control siRNA as indicated . Downregulation of respective was analysed by western blotting as indicated ( WB , three lower panels ) . At 72 h after transfection , were labelled with 32P ‐ orthophosphate for 1 h chased for 2 . 5 h . was separated on a denaturing agarose gel detected by ethidium bromide ( EtBr ) autoradiography after drying of 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 ) signal intensities of 28S 32S rRNA forms were quantified by phosphoimager of 28S : 32S rRNA was calculated . Values represent average of four ( two for siMDN1 ) independent experiments . Error bars indicate s . d .

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(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 species . Endogenous PELP1 was immunoprecipitated from HeLa . In total , 10 % of immunoprecipitated material was analysed by western blotting ( lower panel ) . remaining material was used for isolation cDNA synthesis . cDNA was used as template for PCR amplification using primers within ITS2 / 28S region or GAPDH as a control . Input corresponds to 10 % of lysate . Vertical in 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 extracts . were recovered from fractions by TCA precipitation further analysed by SDS - PAGE 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 depleted of PELP1 , TEX10 , WDR18 , MDN1 or SENP3 . HeLa expressing YFP ‐ RpL27 from a tetracycline ‐ inducible promoter were transfected with indicated siRNAs . After 24 h , expression of YFP ‐ RpL27 was induced 72 h later 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 presence or absence of SV5 ‐ tagged SENP3 in HeLa . Expression of respective 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 transfected with siRNAs as indicated . Depletion of respective 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 were transfected with siRNAs plasmids as indicated . His ‐ SUMO conjugates were recovered on Ni ‐ NTA beads 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 presence of SUMO1 or SUMO2 in HeLa . Expression of respective 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 SUMO were expressed in HeLa 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 by transient transfection . Flag ‐ SUMO2 ‐ expressing were visualized by indirect immunofluorescence . localization of endogenous PELP1 was monitored in Flag ‐ SUMO2 ‐ positive ( green arrowheads ) or untransfected ( 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 were transfected with siRNAs as indicated . After fixation permeabilization , localization of PELP1 , LAS1L , TEX10 or PES1 was monitored by indirect immunofluorescence using 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 were depleted of endogenous PELP1 reconstituted with siRNA resistant constructs expressing either wild ‐ type HA ‐ tagged PELP1 or a Flag ‐ tagged PELP1 - SUMO2 fusion . localization of respective 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 ) localization of LAS1L or SENP3 was analysed in 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 incubated with recombinant E1 E2 enzymes SUMO1 or SUMO2 , respectively , in presence of ATP . In 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 were transfected with siRNAs plasmids as indicated . Expression of respective 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 were transfected with siRNAs plasmids as indicated . His ‐ SUMO2 conjugates were captured on magnetic Ni ‐ NTA beads subjected to western blotting using anti ‐ LAS1L antibodies . Vertical lines indicate removal of irrelevant neighbouring lanes from 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 XRCC4 genomic region encompassing nucleotide substitutions in available members of 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 II - 2 ) control ( CT1 CT2 ) fibroblasts . Each value refers to mean of three independent experiments performed in duplicate . Error bars indicate 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 of total lysates from fibroblasts ( fbs ) of in vitro - synthesized ( i . v . p ) . fl : in vitro - translated full - XRCC4 ; ret . : reticulocyte lysate used for in vitro synthesis ; 225 * : in vitro - translated XRCC4 R225 * truncated ; Ct : lysate of control fbs ; II - 1 II - 2 : lysates of fbs from II - 1 II - 2 . α - XRCC4 ( Abcam , 1 : 1 , 000 ) 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 of total lysates from fibroblasts using α - LIG4 ( GeneTex , 1 : 1 , 000 ) α - SDHA antibodies . Ct : lysate of control fbs ; II - 1 II - 2 : lysates of fbs from II - 1 II - 2 . position of molecular ( MW ) marker 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 foci in XRCC4wt mutant fibroblasts ( XRCC4m / m1 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 1 . 4 0 . 9 foci / nucleus in XRCC4m / m1 XRCC4m / m2 , respectively ) , are mean ± SD of three independent experiments . Percentages of γ - H2AX foci in XRCC4wt XRCC4m / m fibroblasts ( pooled values ) remaining at indicated time - points . Immunofluorescence of γ - H2AX foci in XRCC4wt XRCC4m / m fibroblasts . Data information : * * * P < 0 . 001 ; * * P < 0 . 01 ; * P < 0 . 05 ( XRCC4m / m vs . XRCC4wt ) , two - way ANOVA , Bonferroni post hoc .

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

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

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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 XRCC4m / m irradiated with 0 . 5 Gy of γ - rays were exposed to PARP1 inhibitor 3 ′ - AB or to solvent ( DMSO ) . γ - H2AX foci number is 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 .

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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 ) XRCC4m / m ( B ) from 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 .

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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 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 of m6A / A in purified 18S rRNA 28S rRNA from WT 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 PCR amplification of elongated ligated products of SELECT method for detecting m6A1832 site A1825 site ( for input control ) in 18S rRNA of WT 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 of d3 - m6A / A in 18S rRNA oligos after being incubated with recombinant WT or catalytically mutant mouse METTL5 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 ) AP ( B ) of WT Mettl5 KO mESCs cultured in 2i / Lif medium . Scale bars represent 100 μm 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 of expression of pluripotency genes in WT Mettl5 KO mESCs . Data are represented as mean ± SD from three biological replicates . Student ' s t , two - . 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 of expression of pluripotency markers in WT 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 of expression of pluripotency markers ( POU5F1 , SOX2 NANOG ) in WT Mettl5 KO mESCs , respectively . DAPI ( blue ) was used as a 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 showing dynamic expression patterns of POU5F1 METTL5 during 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 threshold cycles of qPCR ( qPCRCT ) for detecting m6A1832 site in 18S rRNA during indicated time points of EB induction . Data are shown as mean ± SD from three biological replicates . Student ' s t , two - . 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.


C Immunostaining of expression of pluripotency marker POU5F1 ( left ) , endoderm marker FOXA2 ( middle ) neuroectoderm marker NESTIN ( right ) in control Mettl5 KO EBs at different time points , respectively . DAPI ( blue ) was used as a counterstain . Scale bars represent 100 μm . D of percentages of positive shown in ( C ) . Quantitative was on at least three independent experiments . Data are represented as mean ± SD . Student ' s t , two - . * * , P < 0 . 01 ; * * * , P < 0 . 001 .

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C Immunostaining analysis of the expression of pluripotency marker POU5F1(left), endoderm marker FOXA2 (middle) and neuroectoderm marker NESTIN (right) in control and Mettl5 KO EBs at different time points, respectively. DAPI (blue) was used as a nuclear counterstain. Scale bars represent 100 μm. D Quantification of the percentages of positive cells shown in (C). Quantitative analysis was based on at least three independent experiments. Data are represented as mean ± SD. Student's t test, two-tailed. **, P < 0.01; ***, P<0.001.


E RT - qPCR of expressions of pluripotency lineage - specific markers in Day7 EBs . Data are represented as mean ± SD from three biological replicates . Student ' s t , two - . ns , not significant ; * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 .

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E RT-qPCR analysis of the expressions of pluripotency and lineage-specific markers in Day7 EBs. Data are represented as mean ± SD from three biological replicates. Student's t test, two-tailed. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P<0.001.


F Western blot showing pluripotency ( POU5F1 ) three germ layer markers ( FOXA2 , T NESTIN ) expression in Day7 EBs . α - TUBULIN was used as a loading control .

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F Western blot analysis showing pluripotency (POU5F1) and three germ layer markers (FOXA2, T and NESTIN) expression in Day7 EBs. α-TUBULIN was used as a loading control.


A Volcano plot depicting down - regulated ( left ) up - regulated ( right ) genes in Mettl5 KO mESCs compared with WT mESCs in Ribo - seq . Significantly altered genes are defined as those ≥ 1 . 5 - fold change P value < 0 . 05 ( shown in red dots ) . Blue dots represent genes with < 1 . 5 - fold change P value < 0 . 05 . Green dots represent genes with ≥ 1 . 5 - fold change P value ≥ 0 . 05 . Black dots represent genes with ＜ 1 . 5 - fold change P value ≥ 0 . 05 . P values were inferred from two - sided Wald ( DESeq2 ) P value adjustments were made for multiple comparisons . Examples of up - or down - regulated or non - significantly changed genes are annotated in graph .

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A Volcano plot depicting down-regulated (left) and up-regulated (right) genes in Mettl5 KO mESCs compared with WT mESCs in Ribo-seq. Significantly altered genes are defined as those ≥1.5-fold change and P value < 0.05 (shown in red dots). Blue dots represent genes with <1.5-fold change and P value < 0.05. Green dots represent genes with ≥1.5-fold change and P value ≥ 0.05. Black dots represent genes with ＜1.5-fold change and P value ≥ 0.05. P values were inferred from two-sided Wald tests (DESeq2) and P value adjustments were made for multiple comparisons. Examples of up- or down-regulated or non-significantly changed genes are annotated in the graph.


B Track plot showing translational changes of Fbxw7 in Mettl5 KO mESCs compared with controls .

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B Track plot showing translational changes of Fbxw7 in Mettl5 KO mESCs compared with controls.


C RT - qPCR of Fbxw7 mRNA in WT Mettl5 KO during indicated time points course of EB induction . Data are represented as mean ± SD from three biological replicates . Student ' s t , two - . ns , not significant .

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C RT-qPCR analysis of Fbxw7 mRNA levels in WT and Mettl5 KO cells during the indicated time points course of EB induction. Data are represented as mean ± SD from three biological replicates. Student's t test, two-tailed. ns, not significant.


D Western blot showing FBXW7 expression in control Mettl5 KO during indicated time points of EB induction . GAPDH was used as a loading control .

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D Western blot analysis showing FBXW7 expression in control and Mettl5 KO cells during the indicated time points of EB induction. GAPDH was used as a loading control.


E RT - qPCR showing Fbxw7 mRNA in Day7 KO , KO + HA - MT5WT , KO + HA - MT5Mut EBs . Data are represented as mean ± SD from three biological replicates . Student ' s t , two - . ns , not significant .

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E RT-qPCR analysis showing Fbxw7 mRNA levels in Day7 KO, KO+HA-MT5WT, and KO+HA-MT5Mut EBs. Data are represented as mean ± SD from three biological replicates. Student's t test, two-tailed. ns, not significant.


F Western blot showing FBXW7 expression in Day7 KO , KO + HA - MT5WT , KO + HA - MT5Mut EBs . GAPDH was used as a loading control .

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F Western blot analysis showing FBXW7 expression in Day7 KO, KO+HA-MT5WT, and KO+HA-MT5Mut EBs. GAPDH was used as a loading control.


A Western blot showing dynamic expression patterns of FBXW7 c - MYC during 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 FBXW7 and c-MYC during the indicated time points of EB induction. α-TUBULIN was used as a loading control.


B Western blot showing FBXW7 c - MYC expression in control , Mettl5 KO ( KO - 6B ) KO + HA - MT5WT Day7 EBs , respectively . α - TUBULIN was used as a loading control .

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B Western blot analysis showing FBXW7 and c-MYC expression in control, Mettl5 KO (KO-6B) and KO+HA-MT5WT Day7 EBs, respectively. α-TUBULIN was used as a loading control.


D RT - qPCR of expression of pluripotency lineage - specific markers in Day7 FBXW7 - induced EBs at presence or absence of 10 ng / ml Dox . Data are represented as mean ± SD from three biological replicates . Student ' s t , two - . * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 .

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D RT-qPCR analysis of the expression of pluripotency and lineage-specific markers in Day7 FBXW7-induced EBs at the presence or absence of 10 ng/ml Dox. Data are represented as mean ± SD from three biological replicates. Student's t test, two-tailed. *, P < 0.05; **, P < 0.01; ***, P<0.001.


E Immunostaining of expression of pluripotency marker POU5F1 , lineage - specific markers FOXA2 , T NESTIN in Day7 FBXW7 - induced EBs at presence or absence of 10 ng / ml Dox . Scale bars represent 100 μm .

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E Immunostaining analysis of the expression of pluripotency marker POU5F1, lineage-specific markers FOXA2, T and NESTIN in Day7 FBXW7-induced EBs at the presence or absence of 10 ng/ml Dox. Scale bars represent 100 μm.


G RT - qPCR expression of pluripotency lineage - specific markers in Day7 c - MYC - induced EBs at presence or absence of 10 ng / ml Dox . Data are represented as mean ± SD from three biological replicates . Student ' s t , two - . * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 .

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G RT-qPCR analysis the expression of pluripotency and lineage-specific markers in Day7 c-MYC-induced EBs at the presence or absence of 10 ng/ml Dox. Data are represented as mean ± SD from three biological replicates. Student's t test, two-tailed. *, P < 0.05; **, P < 0.01; ***, P<0.001.


H Immunostaining of expression of pluripotency marker POU5F1 , lineage - specific markers FOXA2 , T NESTIN in Day7 c - MYC - induced EBs at presence or absence of 10 ng / ml Dox . Scale bars represent 100 μm .

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H Immunostaining analysis of the expression of pluripotency marker POU5F1, lineage-specific markers FOXA2, T and NESTIN in Day7 c-MYC-induced EBs at the presence or absence of 10 ng/ml Dox. Scale bars represent 100 μm.


C , Validation of AR TRIM33 interaction by co - immunoprecipitation assays . Endogenous AR in LNCaP ( left ) or VCaP ( right ) were immunoprecipitated detected for TRIM33 by immunoblotting . IgG served as a negative control .

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C, Validation of the AR and TRIM33 interaction by co-immunoprecipitation assays. Endogenous AR protein in LNCaP cells (left) or VCaP cells (right) were immunoprecipitated and detected for TRIM33 by immunoblotting. IgG served as a negative control.


D , In vitro GST pull - down assays were performed with bacterially expressed purified GST or GST - tagged AR immobilized onto GSH beads . Immobilized GST beads were used to pull down purified his - MBP ( left ) or his - MBP - tagged TRIM33 ( right ) .

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D, In vitro GST pull-down assays were performed with bacterially expressed and purified GST or GST-tagged AR immobilized onto GSH beads. Immobilized GST protein beads were used to pull down purified his-MBP (left) or his-MBP-tagged TRIM33 (right).


AR - dependent luciferase assays were performed on LNCaP transfected with siTRIM33 or negative control ( NC ) ( A ) , increasing amounts of TRIM33 plasmid ( B ) Western blot shows level of TRIM33 in LNCaP after TRIM33 knockdown ( A ) or overexpression ( B ) . Data information : , data are presented as mean ± SD ( n = 3 independent experiments ) . * P < 0 . 05 ; * * P < 0 . 01 ; ns = not significant ( two - way ANOVA ) .

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AR-dependent luciferase assays were performed on LNCaP cells transfected with siTRIM33 or negative control (NC) (A), increasing amounts of TRIM33 plasmid (B) Western blot analysis shows the protein level of TRIM33 in LNCaP cells after TRIM33 knockdown (A) or overexpression (B). Data information: , data are presented as mean ± SD (n = 3 independent experiments). *P < 0.05; **P < 0.01; ns=not significant (two-way ANOVA).


A - D , AR transcript were measured by RT - qPCR on LNCaP ( A C ) or VCaP ( B D ) transfected with siTRIM33 or TRIM33 expression construct treated with EtOH or DHT . lysates were harvested at 72 h after transfection subjected to western blot with indicated antibodies . Data information : In ( A - D , data are presented as mean ± SD ( n = 3 independent experiments ) . * P < 0 . 05 , * * P < 0 . 01 , ns = not significant ( two - way ANOVA ) .

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A-D, AR transcript levels were measured by RT-qPCR on LNCaP cells (A and C) or VCaP cells (B and D) transfected with siTRIM33 or the TRIM33 expression construct and treated with EtOH or DHT. Protein lysates were harvested at 72 h after transfection and subjected to western blot analysis with the indicated antibodies. Data information: In (A-D, data are presented as mean ± SD (n = 3 independent experiments). * P < 0.05, **P < 0.01, ns=not significant (two-way ANOVA).


E F , LNCaP transfected with siTRIM33 or NC were treated with cycloheximide ( CHX ) . lysates were harvested at indicated time points for western blot . AR were quantified normalized to β - actin then plotted relative to 0 h . Data information : In F , data are presented as mean ± SD ( n = 3 independent experiments ) . * P < 0 . 05 , * * P < 0 . 01 , ns = not significant ( two - way ANOVA ) .

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E and F, LNCaP cells transfected with siTRIM33 or NC were treated with cycloheximide (CHX). Protein lysates were harvested at the indicated time points for western blot analysis. AR protein levels were quantified and normalized to β-actin and then plotted relative to 0 h. Data information: In F, data are presented as mean ± SD (n = 3 independent experiments). * P < 0.05, **P < 0.01, ns=not significant (two-way ANOVA).


G , LNCaP transfected with siTRIM33 or NC were treated with DMSO or MG132 for 8 h . lysates were extracted subjected to western blot with indicated antibodies .

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G, LNCaP cells transfected with siTRIM33 or NC were treated with DMSO or MG132 for 8 h. Protein lysates were extracted and subjected to western blot analysis with the indicated antibodies.


HA - Ub was expressed in LNCaP co - transfected with siTRIM33 ( H ) were treated with MG132 for 4 h before harvesting . Endogenous AR was immunoprecipitated from lysates , interacting were detected by immunoblotting with indicated antibodies .

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HA-Ub was expressed in LNCaP cells co-transfected with siTRIM33 (H) The cells were treated with MG132 for 4 h before harvesting. Endogenous AR was immunoprecipitated from cell lysates, and interacting proteins were detected by immunoblotting with the indicated antibodies.


I , HA - Ub was expressed in LNCaP co - transfected with TRIM33 expression construct ( I ) . were treated with MG132 for 4 h before harvesting . Endogenous AR was immunoprecipitated from lysates , interacting were detected by immunoblotting with indicated antibodies .

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I, HA-Ub was expressed in LNCaP cells co-transfected with the TRIM33 expression construct (I). The cells were treated with MG132 for 4 h before harvesting. Endogenous AR was immunoprecipitated from cell lysates, and interacting proteins were detected by immunoblotting with the indicated antibodies.


LNCaP transiently expressing myc - tagged Skp2 with Flag - TRIM33 were harvested subject to immunoblot with indicated antibodies .

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LNCaP cells transiently expressing myc-tagged Skp2 with Flag-TRIM33 were harvested and subject to immunoblot analysis with the indicated antibodies.


K , LNCaP transiently expressing myc - tagged SPOP with Flag - TRIM33 were harvested subject to immunoblot with indicated antibodies .

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K, LNCaP cells transiently expressing myc-tagged SPOP with Flag-TRIM33 were harvested and subject to immunoblot analysis with the indicated antibodies.


L , shTRIM33 - or shCtrl - LNCaP lines transfected with NC or siSkp2 were harvested 72 h after transfection subjected to immunoblot with indicated antibodies .

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L, shTRIM33- or shCtrl-LNCaP cell lines transfected with NC or siSkp2 were harvested 72 h after transfection and subjected to immunoblot analysis with the indicated antibodies.


M , HEK293T were co - transfected with flag - TRIM33 various myc - tagged AR constructs . AR - FL AR truncated mutants were immunoprecipitated with an anti - myc antibody then subjected to western blot with indicated antibodies .

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M, HEK293T cells were co-transfected with flag-TRIM33 and various myc-tagged AR constructs. AR-FL and AR truncated mutants were immunoprecipitated with an anti-myc antibody and then subjected to western blot analysis with the indicated antibodies.


N , HEK293T were co - transfected with myc - AR - N various flag - tagged TRIM33 constructs . AR - N was immunoprecipitated with an anti - myc antibody then subjected to western blot with indicated antibodies .

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N, HEK293T cells were co-transfected with myc-AR-N and various flag-tagged TRIM33 constructs. AR-N was immunoprecipitated with an anti-myc antibody and then subjected to western blot analysis with the indicated antibodies.


O , LNCaP were transfected with various flag - tagged TRIM33 constructs . AR - associated were immunoprecipitated with an anti - AR antibody then subjected to western blot with indicated antibodies .

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O, LNCaP cells were transfected with various flag-tagged TRIM33 constructs. AR-associated proteins were immunoprecipitated with an anti-AR antibody and then subjected to western blot analysis with the indicated antibodies.


P , LNCaP were co - transfected with myc - Skp2 various flag - TRIM33 constructs . AR other were detected by western blot with indicated antibodies .

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P, LNCaP cells were co-transfected with myc-Skp2 and various flag-TRIM33 constructs. AR and other proteins were detected by western blot analysis with the indicated antibodies.


Q , AR pull - down was performed on extracts from MG132 - treated HEK293T overexpressing myc - AR , HA - Ub , myc - Skp2 , various flag - tagged TRIM33 constructs . AR ubiquitination specific were detected by western blot with indicated antibodies .

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Q, AR pull-down was performed on protein extracts from MG132-treated HEK293T cells overexpressing myc-AR, HA-Ub, myc-Skp2, and various flag-tagged TRIM33 constructs. AR ubiquitination and specific protein levels were detected by western blot analysis with the indicated antibodies.


A , TRIM33 mRNA expression were analyzed in normal samples from multiple publicly available cohorts . A two - sided Wilcoxon signed - rank was used for TCGA Prostate , Ding 2016 , Jing 2020 datasets , while a two - sided Wilcoxon rank - sum was used for other datasets . Measurements shown in box plots are first third quartiles are split by medians , whiskers extending a 1 . 5 - fold interquartile range beyond box .

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A, TRIM33 mRNA expression levels were analyzed in normal and tumor samples from multiple publicly available patient cohorts. A two-sided Wilcoxon signed-rank test was used for the TCGA Prostate, Ding 2016, and Jing 2020 datasets, while a two-sided Wilcoxon rank-sum test was used for the other datasets. Measurements shown in box plots are the first and third quartiles and are split by the medians, whiskers extending a 1.5-fold interquartile range beyond the box.


B C , A human PCa progression tissue microarray was used for immunohistochemistry . Representative are shown in B . TRIM33 by Gleason Scores is summarized in histogram . Scale bar : 100 μm .

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B and C, A human PCa progression tissue microarray was used for immunohistochemistry staining. Representative staining images are shown in B. TRIM33 staining by Gleason Scores is summarized in the histogram. Scale bar: 100 μm.


A , AR - TRIM33 coactivated gene signature was compared with two PCa - derived AR gene signatures in multiple publicly available datasets .

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A, The AR-TRIM33 coactivated gene signature was compared with two PCa patient-derived AR gene signatures in multiple publicly available patient datasets.


B , expression of gene signature in normal samples was examined in same cohorts used in A . A two - sided Wilcoxon signed - rank was used for TCGA Prostate , Ding 2016 , Jing 2020 datasets , while a two - sided Wilcoxon rank - sum was used for other datasets . Measurements shown in box plots are first third quartiles are split by medians , whiskers extending a 1 . 5 - fold interquartile range beyond box .

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B, The expression of the gene signature in normal and tumor samples was examined in the same cohorts used in A. A two-sided Wilcoxon signed-rank test was used for the TCGA Prostate, Ding 2016, and Jing 2020 datasets, while a two-sided Wilcoxon rank-sum test was used for the other datasets. Measurements shown in box plots are the first and third quartiles and are split by the medians, whiskers extending a 1.5-fold interquartile range beyond the box.


C , Hierarchical clustering of PCa from TCGA Prostate dataset ( n = 480 ) resulted in two distinct clusters indicated in orange ( high ) blue ( low ) on average expression of our 36 - gene signature .

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C, Hierarchical clustering of PCa patients from the TCGA Prostate dataset (n = 480) resulted in two distinct clusters indicated in orange (high) and blue (low) based on the average expression of our 36-gene signature.


E , Kaplan - Meier showing recurrence - free survival of with low high expression of 36 - gene signature ( p - value , log - rank ) .

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E, Kaplan-Meier analysis showing the recurrence-free survival of patients with low and high expression of the 36-gene signature (p-value, log-rank test).


J , Western blot of AR TRIM3 in tissues from H .

J, Western blot analysis of AR and TRIM3 protein levels in tumor tissues from H.


( A B ) FACS - sorted T populations Monocyte - derived Macrophages were treated with indicated stimuli : Nigericin ( Nig . 4h ) , NeedleTox ( 4h ) , Val - boroPro ( VbP 22h ) , ABT737 / S63845 ( A / S 22h ) . When indicated , were primed with LPS for 2 hours prior to stimulation . When indicated , MCC950 was added to media 30 minutes prior to addition of Nigericin . LDH - activity ( A ) IL - 1β IL - 18 ( B C , respectively ) in supernatant were determined by LDH cytotoxicity ELISA respectively . data points ± from three independent donors are shown . Statistics indicate significance by two - way ANOVA : * * * p ≤ 0 . 001 ; * * p ≤ 0 . 01 ; * p ≤ 0 . 05 ; ns , not significant . P - values were corrected for multiple comparisons ( Dunnett ) .

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(A and B) FACS-sorted T cell populations and Monocyte-derived Macrophages were treated with the indicated stimuli: Nigericin (Nig. 4h), NeedleTox (4h), Val-boroPro (VbP 22h), ABT737/S63845 (A/S 22h). When indicated, cells were primed with LPS for 2 hours prior to stimulation. When indicated, MCC950 was added to the media 30 minutes prior to the addition of Nigericin. LDH-activity (A) and IL-1β and IL-18 concentration (B and C, respectively) in the supernatant were determined by LDH cytotoxicity assay and ELISA respectively. Individual data points ± SEM from three independent donors are shown. Statistics indicate significance by two-way ANOVA: ***p ≤ 0.001; **p ≤ 0.01; *p ≤ 0.05; ns, not significant. P-values were corrected for multiple comparisons (Dunnett).


A ) Macrophages CD4 T from same donor were subjected to indicated treatments morphologic changes as well as PI - uptake were monitored by live - imaging microscopy using a 25x objective . Representative from indicated time points are shown . Cyan color coding is used for PI - signal . One donor out of two is shown . ( B ) Representative were acquired with a 63x objective at end of experiment shown in ( A ) at 16 hours . One donor out of two is shown . Scale bars : 25 µm .

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A) Macrophages and CD4 T cells from the same donor were subjected to the indicated treatments and morphologic changes as well as PI-uptake were monitored by live-cell imaging microscopy using a 25x objective. Representative images from indicated time points are shown. Cyan color coding is used for the fluorescent PI-signal. One donor out of two is shown. (B) Representative images were acquired with a 63x objective at the end of the experiment shown in (A) at 16 hours. One donor out of two is shown. Scale bars: 25 µm.