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 =

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

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 .

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

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 .

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

(A, B) 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 .

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

(C) 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 .

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

(D-F) 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 .

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

(A, B) 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 .

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

(C) 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 .

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

(D) 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 .

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

(E) 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 .

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

(A-C) 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 .

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

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

(D) 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 .

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

(E) 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 .

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

(F) 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 .

(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 .

(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 .

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

(C) 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 .

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

(D) 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 .

(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 .

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

(A) 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 .

O O O O O O O O O O O O O O O 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-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 .

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

(E) 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 .

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

(F) 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 .

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

(A) 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 .

(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 ) .

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

(C) 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 .

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 .

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

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 .

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

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 .

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

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 .

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

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 .

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

A, B 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 .

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

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 .

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

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 .

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

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 .

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

C 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 .

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 .

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

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 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 .

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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 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 .

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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 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 .

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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 ) 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 .

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

A 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 .

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


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 .

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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 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 .

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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 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 .

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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 analysis showing FBXW7 expression in Day7 KO , KO + HA - MT5WT , and 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 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 .

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 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 .

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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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 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 ) .

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 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 ) .

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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 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 ) .

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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 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 ) .

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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 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 .

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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 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 .

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

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 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 .

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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 cells transiently expressing myc - tagged Skp2 with Flag - TRIM33 were harvested and subject to immunoblot analysis with the 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 cells transiently expressing myc - tagged SPOP with Flag - TRIM33 were harvested and subject to immunoblot analysis with the 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 cell lines transfected with NC or siSkp2 were harvested 72 h after transfection and subjected to immunoblot analysis with the 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 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 .

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

M, 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

N, 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 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 .

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

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 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 .

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

P, 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 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 .

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 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 .

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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 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 .

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

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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 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 .

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

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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 analysis of AR and TRIM3 protein levels in tumor tissues from H .

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


( 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 ) .

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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 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 .

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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.


( C ) CD4 T cells were treated with VbP or ABT737 / S63845 . Lysate and supernatant samples were collected at the indicated time points . Samples were analyzed by immunoblotting . αSS and αLS indicate the use of a small subunit or large subunit - specific caspase - 1 antibody respectively . ( Lys = lysate , SN = supernatant , FL = full length ) . One representative experiment out of three is shown .

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(C) CD4 T cells were treated with VbP or ABT737/S63845. Lysate and supernatant samples were collected at the indicated time points. Samples were analyzed by immunoblotting. αSS and αLS indicate the use of a small subunit or large subunit-specific caspase-1 antibody respectively. (Lys = lysate, SN = supernatant, FL = full length). One representative experiment out of three is shown.


( A ) Gene expression profile of 15 inflammasome - related genes in human T cell and monocyte subsets . TPM values were retrieved from a public RNASeq dataset and log2 transformed . Both , rows and columns are clustered using euclidean distance and complete linkage . ( Tfh , T follicular helper cells ; Tregs , T regulatory cells ; Th , T helper cells ; CM , central memory T cells ; EM , effector memory T cells ; TE , terminal effector T cells ; CD14 + C16 - , classical monocytes ; CD14 + CD16 + , intermediate monocytes ; CD14 - CD16 + , non - classical monocytes ) .

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(A) Gene expression profile of 15 inflammasome-related genes in human T cell and monocyte subsets. TPM values were retrieved from a public RNASeq dataset and log2 transformed. Both, rows and columns are clustered using euclidean distance and complete linkage. (Tfh, T follicular helper cells; Tregs, T regulatory cells; Th, T helper cells; CM, central memory T cells; EM, effector memory T cells; TE, terminal effector T cells; CD14+ C16-, classical monocytes; CD14+ CD16+, intermediate monocytes; CD14- CD16+, non-classical monocytes).


( B ) Immunoblotting of CD4 T cells and MDMs treated with Pam3CSK4 for 6 hours . * indicates an unspecific band . One representative experiment out of three is shown .

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(B) Immunoblotting of CD4 T cells and MDMs treated with Pam3CSK4 for 6 hours. * indicates an unspecific band. One representative experiment out of three is shown.


( C ) Immunoblotting of polyclonal CD4 T cells targeted with two gRNAs for each indicated target gene . One representative experiment out of three is shown .

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(C) Immunoblotting of polyclonal CD4 T cells targeted with two gRNAs for each indicated target gene. One representative experiment out of three is shown.


( D ) CD4 T cells from the indicated polyclonal gene targeting approach were treated with the indicated stimuli for 18 hours . Individual data points ± SEM from three independent donors are shown . Statistics indicate significance by two - way ANOVA : * * * p ≤ 0 . 001 ; ns , not significant . P - values were corrected for multiple comparisons ( Dunnett ) .

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(D) CD4 T cells from the indicated polyclonal gene targeting approach were treated with the indicated stimuli for 18 hours. Individual data points ± SEM from three independent donors are shown. Statistics indicate significance by two-way ANOVA: ***p ≤ 0.001; ns, not significant. P-values were corrected for multiple comparisons (Dunnett).


( B ) MiSeq analysis of the indicated gene targeting approaches from samples collected at the indicated time points post editing . DPP9 and DPP8 were targeted with one gRNA / target gene , CARD8 was targeted with two gRNAs . On the day of nucleofection , T cells were activated with human T activator CD3 / CD28 beads and expanded in the presence of IL - 2 . On day 10 , cells were switched to IL - 7 / IL - 15 containing medium . ( C ) Fraction of wildtype and in - frame reads are depicted . One representative experiment out of three is shown .

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(B) MiSeq analysis of the indicated gene targeting approaches from samples collected at the indicated time points post editing. DPP9 and DPP8 were targeted with one gRNA/target gene, CARD8 was targeted with two gRNAs. On the day of nucleofection, T cells were activated with human T activator CD3/CD28 beads and expanded in the presence of IL-2. On day 10, cells were switched to IL-7/IL-15 containing medium. (C) Fraction of wildtype and in-frame reads are depicted. One representative experiment out of three is shown.


( A ) CD4 T cells were activated with human T activator CD3 / CD28 beads ( bead : cell ratio 1 : 5 ) for 4 days . Controls were kept resting in the presence of IL - 7 and IL - 15 . On day 4 cells were stimulated as indicated for 18 hours and cytotoxicity was assessed by LDH - activity in the supernatant . Individual data points ± SEM from four independent donors . Statistics indicate significance by two - way ANOVA : * * * p ≤ 0 . 001 ; ns , not significant . P - values were corrected for multiple comparisons ( Sidak ) .

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(A) CD4 T cells were activated with human T activator CD3/CD28 beads (bead:cell ratio 1:5) for 4 days. Controls were kept resting in the presence of IL-7 and IL-15. On day 4 cells were stimulated as indicated for 18 hours and cytotoxicity was assessed by LDH-activity in the supernatant. Individual data points ± SEM from four independent donors. Statistics indicate significance by two-way ANOVA: ***p ≤ 0.001; ns, not significant. P-values were corrected for multiple comparisons (Sidak).


( B ) Immunoblotting of CD4 T cell lysates treated as in ( A ) for 3 to 4 days . * indicates an unspecific band . ( C ) Quantification of immunoblots shown in ( B ) and additional donors ( n = 9 for all but GSDMD , GSDMD n = 8 , for one donor quantification of the GSDMD band was considered unreliable due to uneven substrate distribution ) . Individual data points ± SEM are shown . Statistics indicate significance by one - way ANOVA of log2 - transformed fold change values of band intensities ( activated / non - activated T cells ) normalized to β - Actin : * * * p ≤ 0 . 001 ; * p ≤ 0 . 05 ; ns , not significant . P - values were corrected for multiple comparisons ( Sidak ) . All band intensity ratios were tested against β - Actin . CARD8 full length was additionally tested against CARD8 cleaved .

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(B) Immunoblotting of CD4 T cell lysates treated as in (A) for 3 to 4 days. * indicates an unspecific band. (C) Quantification of immunoblots shown in (B) and additional donors (n= 9 for all but GSDMD, GSDMD n=8, for one donor quantification of the GSDMD band was considered unreliable due to uneven substrate distribution). Individual data points ± SEM are shown. Statistics indicate significance by one-way ANOVA of log2-transformed fold change values of band intensities (activated/non-activated T cells) normalized to β-Actin: ***p ≤ 0.001; *p ≤ 0.05; ns, not significant. P-values were corrected for multiple comparisons (Sidak). All band intensity ratios were tested against β-Actin. CARD8 full length was additionally tested against CARD8 cleaved.


A , B Data were normalised using the control group and are represented as fold - change ( FC ) of the mean fluorescence intensity ( MFI ) . Macrophage cells were obtained from human donors ( n = 4 biological replicas ) and incubated with medium ( CON ) or recombinant human IL - 10 ( hIL - 10r ) , MPN wild - type ( WT ) or MPN expressing IL - 10 ORF ( WT _ IL10 ) . Data are represented as mean ± Standard Error of the Mean ( SEM ) . Statistical analysis was performed using one - way ANOVA + post - hoc Tukey multiple comparison test ( * P < 0 . 05 ; * * P < 0 . 001 , * * * P < 0 . 0001 ) .

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A, B Data were normalised using the control group and are represented as fold-change (FC) of the mean fluorescence intensity (MFI). Macrophage cells were obtained from human donors (n = 4 biological replicas) and incubated with medium (CON) or recombinant human IL-10 (hIL-10r), MPN wild-type (WT) or MPN expressing IL-10 ORF (WT_IL10). Data are represented as mean ± Standard Error of the Mean (SEM). Statistical analysis was performed using one-way ANOVA + post-hoc Tukey multiple comparison test (*P < 0.05; **P < 0.001, ***P < 0.0001).


( A ) EC - 50 ( molar , M ) for human IL - 10 recombinant protein ( hIL - 10r ) and IL - 10 WT ( IL - 10 ORF ) and different variants ( MutM , hIL - 10r , Mut1 , Mut2 , Mut3 , MutSC1 , and MutSC2 ) expressed by M . pneumoniae . Data are represented as mean ± SD . Statistical comparison was done by One - way ANOVA + post - hoc Bonferroni multiple comparison test , using the IL - 10 ORF condition as a reference ( * P < 0 . 05 ) .

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(A) EC-50 (molar, M) for human IL-10 recombinant protein (hIL-10r) and IL-10 WT (IL-10 ORF) and different variants (MutM, hIL-10r, Mut1, Mut2, Mut3, MutSC1, and MutSC2) expressed by M. pneumoniae. Data are represented as mean ± SD. Statistical comparison was done by One-way ANOVA + post-hoc Bonferroni multiple comparison test, using the IL-10 ORF condition as a reference (*P < 0.05).


( B ) Fold - change ( FC ) in expression levels ( fg / CFU ) of IL - 10 variants Mut3 and MutSC1 secreted to the medium normalised by expression level of IL - 10 ORF ( ORF ) . Statistical comparison of mean ± SD was performed by One - way ANOVA + Tukey post - hoc test ( * P < 0 . 05 ) .

(B) Fold-change (FC) in expression levels (fg/CFU) of IL-10 variants Mut3 and MutSC1 secreted to the medium normalised by expression level of IL-10 ORF (ORF). Statistical comparison of mean ± SD was performed by One-way ANOVA + Tukey post-hoc test (*P < 0.05).


( C , D ) Average and SD values for the FC in the relative EC - 50 values determined by flow cytometry analysis of phosphorylated STAT3 after a 20 - min exposure of the BlaER1 ( C ) or HAFTL ( D ) cell lines to IL - 10 ORF ( reference ) or the mutant MutSC1 or MutSC2 Numbers indicate the average FC ± SD .

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(C, D) Average and SD values for the FC in the relative EC-50 values determined by flow cytometry analysis of phosphorylated STAT3 after a 20-min exposure of the BlaER1 (C) or HAFTL (D) cell lines to IL-10 ORF (reference) or the mutant MutSC1 or MutSC2 Numbers indicate the average FC ± SD.


C57Bl / 6 were infected with 107 CFUs of WT or CV8 strains and sacrificed at 2 days post - infection ( dpi ) or 4 - dpi ( n ≥ 3 biological replicas / group ) . ( A ) Average ± SD bacterial loads recovered of the WT ( circle ) and CV8 ( square ) strains in mouse lungs at 2 - dpi or 4 - dpi . Data are shown as log10 CFU / lung homogenate .

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C57Bl/6 were infected with 107 CFUs of WT or CV8 strains and sacrificed at 2 days post-infection (dpi) or 4-dpi (n ≥ 3 biological replicas/group). (A) Average ± SD bacterial loads recovered of the WT (circle) and CV8 (square) strains in mouse lungs at 2-dpi or 4-dpi. Data are shown as log10 CFU/ lung homogenate.


C57Bl / 6 were infected with 107 CFUs of WT or CV8 strains and ( B ) Inflammatory profile of mouse lungs inoculated with WT ( dark blue ) , CV8 ( light blue ) , or PBS ( grey ) . Gene expression was analysed by RT - qPCR using gapdh as an endogenous control ( see Materials and Methods ) . Data are shown as average ± SD of fold - change ( FC ) in mRNA expression ( One - way ANOVA + Tukey post - hoc test ; * P < 0 . 05 ) .

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C57Bl/6 were infected with 107 CFUs of WT or CV8 strains and (B) Inflammatory profile of mouse lungs inoculated with WT (dark blue), CV8 (light blue), or PBS (grey). Gene expression was analysed by RT-qPCR using gapdh as an endogenous control (see Materials and Methods). Data are shown as average ± SD of fold-change (FC) in mRNA expression (One-way ANOVA + Tukey post-hoc test; *P < 0.05).


C57Bl / 6 were infected with 107 CFUs of WT or CV8 strains and sacrificed at 2 days post - infection ( dpi ) or 4 - dpi ( n ≥ 3 biological replicas / group ) . ( C ) Histological findings of mouse lung samples at the analysed time points . Left , plots representing the quantitative evaluation ( score : 0 - 5 ) of alveolar infiltrate ( top panel ) and peribronquial / olar infiltrate ( FC ) ( bottom panel ) ( see Materials and Methods ) . Parameters were normalised using the average of the PBS group ( FC = sample value / average PBS ; FC control group , ~ 1 ) . Data are shown as average ± SD of FC ( one - way ANOVA + Tukey post - hoc test ) ( * P < 0 . 05 ) . Right , representative images of lungs stained with hematoxylin - eosin ( the line represents 250 μm ) .

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C57Bl/6 were infected with 107 CFUs of WT or CV8 strains and sacrificed at 2 days post-infection (dpi) or 4-dpi (n ≥ 3 biological replicas/group). (C) Histological findings of mouse lung samples at the analysed time points. Left, plots representing the quantitative evaluation (score: 0-5) of alveolar infiltrate (top panel) and peribronquial/olar infiltrate (FC) (bottom panel) (see Materials and Methods). Parameters were normalised using the average of the PBS group (FC = sample value/average PBS; FC control group, ~1). Data are shown as average ± SD of FC (one-way ANOVA + Tukey post-hoc test) (*P < 0.05). Right, representative images of lungs stained with hematoxylin-eosin (the line represents 250 μm).


C57Bl / 6 were infected with 107 CFUs of WT or CV8 strains and ( D ) IL - 10 secretion by the MPN WT or CV8 strain coding for hIL - 10 WT ( ORF ) , MutSC1 , or MutSC2 . Data are expressed as the mean ± SD of IL - 10 supernatant concentration ( μg / mL ) by biomass ( protein content in μg / mL ) ( n = 3 biological replicas ) .

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C57Bl/6 were infected with 107 CFUs of WT or CV8 strains and (D) IL-10 secretion by the MPN WT or CV8 strain coding for hIL-10 WT (ORF), MutSC1, or MutSC2. Data are expressed as the mean ± SD of IL-10 supernatant concentration (μg/mL) by biomass (protein content in μg/mL) (n=3 biological replicas).


( B ) CFUs of PAO1 ( left ) or Mycoplasma strains ( right ) recovered from lung samples . Data are shown as mean ± SD of Log10 CFU / lung homogenate . Two replicates were performed and individual symbols are shown ( experiment 1 , circle ; experiment 2 , square ) .

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(B) CFUs of PAO1 (left) or Mycoplasma strains (right) recovered from lung samples. Data are shown as mean ± SD of Log10 CFU/lung homogenate. Two replicates were performed and individual symbols are shown (experiment 1, circle; experiment 2, square).


( C ) Fold - change ( FC ) of mRNA gene expression of inflammatory markers determined by qPCR . The 2 - ΔΔCt method was used to normalise the values , using gapdh as endogenous control Data are shown as average ± SD of FC . Statistical analyses were performed using unpaired t - test ( * P < 0 . 05 ; * * P < 0 . 01 * * * ; P < 0 . 001 , * * * * P < 0 . 0001 ) . Two replicates were performed and individual symbols are shown ( Experiment 1 , circle ; Experiment 2 , square ) .

(C) Fold-change (FC) of mRNA gene expression of inflammatory markers determined by qPCR. The 2-ΔΔCt method was used to normalise the values, using gapdh as endogenous control Data are shown as average ± SD of FC. Statistical analyses were performed using unpaired t-test (* P < 0.05; ** P <0.01***; P <0.001, **** P <0.0001). Two replicates were performed and individual symbols are shown (Experiment 1, circle; Experiment 2, square).


( D ) Quantitative analysis of immunochemistry ( IHC ) against neutrophil elastase ( NE ) of lung samples . Data are represented as the mean ± SD of percentage of positive cells , calculated as follows : % = 100 × ( positive cells / positive cells + negative cells ) ( One - Way ANOVA + Tukey post - hoc test , * P < 0 . 05 ) . ( E ) Representative images of IHC staining of lung samples against NE are shown . Arrows indicate NE - positive cells . Scale bar size : 50 µm .

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(D) Quantitative analysis of immunochemistry (IHC) against neutrophil elastase (NE) of lung samples. Data are represented as the mean ± SD of percentage of positive cells, calculated as follows: % = 100 × (positive cells/positive cells + negative cells) (One-Way ANOVA + Tukey post-hoc test, * P <0.05). (E) Representative images of IHC staining of lung samples against NE are shown. Arrows indicate NE-positive cells. Scale bar size: 50 µm.


( a ) Ultrastructural evidence of autophagy induced by depletion of p53 with a specific siRNA or pharmacological inhibition of p53 with PFT - α in HCT116 cells .

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(a) Ultrastructural evidence of autophagy induced by depletion of p53 with a specific siRNA or pharmacological inhibition of p53 with PFT-α in HCT116 cells.


( b ) Autophagy induced by PFT - α , p53 knockdown , or p53 knockout in HCT116 cells . The number of autophagosomes and autolysosomes was determined for at least 50 cells in 3 independent experiments ( mean ± s . e . m . ; * P 0 . 05 ) . Culture in nutrient - free ( NF ) conditions was used as a positive control .

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(b) Autophagy induced by PFT-α, p53 knockdown, or p53 knockout in HCT116 cells. The number of autophagosomes and autolysosomes was determined for at least 50 cells in 3 independent experiments (mean ± s.e.m.; *P 0.05). Culture in nutrient-free (NF) conditions was used as a positive control.


( c ) Effect of p53 on the maturation of LC3 . Immunoblots are shown for wild - type ( WT ) or p53 − / − HCT116 ( n = 3 ) .

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(c) Effect of p53 on the maturation of LC3. Immunoblots are shown for wild-type (WT) or p53−/− HCT116 (n = 3).


( d ) GFP - LC3 puncta induced by PFT - α , p53 knockdown or knockout . WT and p53 − / − HCT116 cells were transfected with control or p53 - specific siRNAs , re - transfected with a GFP - LC3 plasmid , cultured in complete medium for 24 h and kept for 6 h in the presence or absence of 30 μM PFT - α . The percentage of cells showing accumulation of GFP - LC3 in puncta ( GFP - LC3vac ) is reported ( mean ± s . d . , n = 3 ; * P 0 . 05 ) .

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(d) GFP-LC3 puncta induced by PFT-α, p53 knockdown or knockout. WT and p53−/− HCT116 cells were transfected with control or p53-specific siRNAs, re-transfected with a GFP-LC3 plasmid, cultured in complete medium for 24 h and kept for 6 h in the presence or absence of 30 μM PFT-α. The percentage of cells showing accumulation of GFP-LC3 in puncta (GFP-LC3vac) is reported (mean ± s.d., n = 3; *P 0.05).


( e ) GFP - LC3 puncta in p53 − / − MEF , compared with WT MEF transfected with GFP - LC3 ( mean ± s . d . , n = 3 ; * P 0 . 05 ) .

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(e) GFP-LC3 puncta in p53−/− MEF, compared with WT MEF transfected with GFP-LC3 (mean ± s.d., n = 3; *P 0.05).


( f ) GFP - LC3 dots in tissues from p53 + / + or p53 − / − mice expressing a GFP - LC3 transgene ( mean ± s . d . ; * P 0 . 05 ) , without or after 24 h of starvation . The number of GFP - LC3 dots per area was determined for a minimum of 4 fields ( at × 400 magnification for 4 slides per animal , 3 animals per condition ) .

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(f) GFP-LC3 dots in tissues from p53+/+ or p53−/− mice expressing a GFP-LC3 transgene (mean ± s.d.; *P 0.05), without or after 24 h of starvation. The number of GFP-LC3 dots per area was determined for a minimum of 4 fields (at ×400 magnification for 4 slides per animal, 3 animals per condition).


( g ) TEM pictures of p53 − / − and p53 + / − mouse livers . p53 − / − hepatocytes carry approximately 3 autophagosomes , p53 + / − controls approximately 1 per cell .

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(g) TEM pictures of p53−/− and p53+/− mouse livers. p53−/− hepatocytes carry approximately 3 autophagosomes, p53+/− controls approximately 1 per cell.


( h ) Modulation of DsRed : : LGG - 1 levels by the p53 orthologue CEP - 1 in C . elegans . Representative pictures are shown for WT animals and for nematodes carrying a cep - 1 deletion allele ( gk138 ) . The count of puncta per cell was 6 . 9 ± 3 . 3 for WT , 52 . 2 ± 9 . 4 for cep - 1 ( gk138 ) embryos ( mean ± s . e . m . for 20 individuals , 5 cells per embryo ) . The quantification of DsRed : : LGG - 1 puncta is shown for the indicated genetic backgrounds and conditions ( dots represent pixel intensity for individual embryos ) .

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(h) Modulation of DsRed::LGG-1 levels by the p53 orthologue CEP-1 in C. elegans. Representative pictures are shown for WT animals and for nematodes carrying a cep-1 deletion allele (gk138). The count of puncta per cell was 6.9 ± 3.3 for WT, 52.2 ± 9.4 for cep-1 (gk138) embryos (mean ± s.e.m. for 20 individuals, 5 cells per embryo). The quantification of DsRed::LGG-1 puncta is shown for the indicated genetic backgrounds and conditions (dots represent pixel intensity for individual embryos).


( a ) PFT - α or p53 siRNA - induced GFP - LC3 puncta in WT HCT116 cells transfected with siRNAs to deplete the essential autophagy gene products Atg5 , Atg10 , Atg12 , Beclin 1 or hVps34 . Culture in nutrient - free ( NF ) conditions was used as a control of autophagy induction .

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(a) PFT-α or p53 siRNA-induced GFP-LC3 puncta in WT HCT116 cells transfected with siRNAs to deplete the essential autophagy gene products Atg5, Atg10, Atg12, Beclin 1 or hVps34. Culture in nutrient-free (NF) conditions was used as a control of autophagy induction.


( b ) PFT - α or p53 siRNA - mediated GFP - LC3 puncta in MEFs transfected with siRNAs specific for mouse Atg5 or Beclin 1 .

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(b) PFT-α or p53 siRNA-mediated GFP-LC3 puncta in MEFs transfected with siRNAs specific for mouse Atg5 or Beclin 1.


( c ) Requirement of Beclin 1 expression for autophagy induction by p53 depletion in MCF7 cells . MCF7 cells stably transfected with a tetracycline - repressible Beclin 1 construct were maintained in conditions that prevent or allow Beclin 1 expression , transfected with GFP - LC3 and subjected to p53 knockdown before quantification of GFP - LC3 puncta . Data in a - c are mean ± s . d . of 3 independent experiments .

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(c) Requirement of Beclin 1 expression for autophagy induction by p53 depletion in MCF7 cells. MCF7 cells stably transfected with a tetracycline-repressible Beclin 1 construct were maintained in conditions that prevent or allow Beclin 1 expression, transfected with GFP-LC3 and subjected to p53 knockdown before quantification of GFP-LC3 puncta. Data in a-c are mean ± s.d. of 3 independent experiments.


( d - f ) Effect of bafilomycin A1 ( BafA1 ) on PFT - α - induced GFP - LC3 puncta . GFP - LC3 - expressing HeLa cells were treated with PFT - α and / or BafA1 and then immunostained for Lamp - 2 to observe the colocalization between GFP - LC3 and Lamp - 2 . Representative confocal microphotographs are shown together with the profiles of colocalization ( d ) within the area of interest , indicated by the orientation of the arrow . Percentage overlap was plotted for control cells and cells cultured in the presence of PFT - α and / or BafA1 after 6 h ( d , e ) . Columns represent the percentage of colocalization of GFP - LC3 and Lamp - 2 ( mean ± s . d . ; * P 0 . 05 ) , quantified for at least 50 cells for each condition . The kinetics of GFP - LC3 redistribution was quantified by conventional fluorescence microscopy ( mean ± s . d . , 3 experiments , f ) .

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(d-f) Effect of bafilomycin A1 (BafA1) on PFT-α-induced GFP-LC3 puncta. GFP-LC3-expressing HeLa cells were treated with PFT-α and/or BafA1 and then immunostained for Lamp-2 to observe the colocalization between GFP-LC3 and Lamp-2. Representative confocal microphotographs are shown together with the profiles of colocalization (d) within the area of interest, indicated by the orientation of the arrow. Percentage overlap was plotted for control cells and cells cultured in the presence of PFT-α and/or BafA1 after 6 h (d, e). Columns represent the percentage of colocalization of GFP-LC3 and Lamp-2 (mean ± s.d.; *P 0.05), quantified for at least 50 cells for each condition. The kinetics of GFP-LC3 redistribution was quantified by conventional fluorescence microscopy (mean ± s.d., 3 experiments, f).


( d - f ) Effect of bafilomycin A1 ( BafA1 ) on PFT - α - induced GFP - LC3 puncta . GFP - LC3 - expressing HeLa cells were treated with PFT - alpha and / or BafA1 and then immunostained for Lamp - 2 to observe the colocalization between GFP - LC3 and Lamp - 2 . Representative confocal microphotographs are shown together with the profiles of colocalization ( d ) within the area of interest , indicated by the orientation of the arrow . Percentage overlap was plotted for control cells and cells cultured in the presence of PFT - α and / or BafA1 after 6 h ( d , e ) . Columns represent the percentage of colocalization of GFP - LC3 and Lamp - 2 ( mean ± s . d . ; * P 0 . 05 ) , quantified for at least 50 cells for each condition . The kinetics of GFP - LC3 redistribution was quantified by conventional fluorescence microscopy ( mean ± s . d . , 3 experiments , f ) .

(d-f) Effect of bafilomycin A1 (BafA1) on PFT-α-induced GFP-LC3 puncta. GFP-LC3-expressing HeLa cells were treated with PFT-alpha and/or BafA1 and then immunostained for Lamp-2 to observe the colocalization between GFP-LC3 and Lamp-2. Representative confocal microphotographs are shown together with the profiles of colocalization (d) within the area of interest, indicated by the orientation of the arrow. Percentage overlap was plotted for control cells and cells cultured in the presence of PFT-α and/or BafA1 after 6 h (d, e). Columns represent the percentage of colocalization of GFP-LC3 and Lamp-2 (mean ± s.d.; *P 0.05), quantified for at least 50 cells for each condition. The kinetics of GFP-LC3 redistribution was quantified by conventional fluorescence microscopy (mean ± s.d., 3 experiments, f).


( g ) Immunoblot detection of LC3 - I / II in cells treated with PFT - α and / or leupeptin in WT HCT116 cells ( n = 3 ) .

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(g) Immunoblot detection of LC3-I/II in cells treated with PFT-α and/or leupeptin in WT HCT116 cells (n = 3).


( a ) Immunoblot detection of the phosphorylation status of AMPKα , ACCα , TSC2 and p70S6K in HCT116 cells . Hypophosphorylation of p70S6K and hyperphosphorylation of AMPKα , ACCα and TSC2 was detected in p53 − / − HCT116 , compared with WT HCT116 cells after culture in complete medium ( n = 5 ) .

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(a) Immunoblot detection of the phosphorylation status of AMPKα, ACCα, TSC2 and p70S6K in HCT116 cells. Hypophosphorylation of p70S6K and hyperphosphorylation of AMPKα, ACCα and TSC2 was detected in p53−/− HCT116, compared with WT HCT116 cells after culture in complete medium (n = 5).


( b ) Quantification of GFP - LC3vac cells in WT and p53 − / − HCT116 cells transfected with siRNAs specific for the catalytic α1 and α2 subunits of AMPK ( mean ± s . d . of 3 independent experiments ; * P 0 . 05 ) . The inset in b demonstrates the efficiency of siRNA - mediated downregulation of AMPKα1 and AMPKα2 , as assessed by immunoblot analysis ( n = 3 ) . Note that the antibody recognizes both AMPKα1 and AMPKα2 .

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(b) Quantification of GFP-LC3vac cells in WT and p53−/− HCT116 cells transfected with siRNAs specific for the catalytic α1 and α2 subunits of AMPK (mean ± s.d. of 3 independent experiments; *P 0.05). The inset in b demonstrates the efficiency of siRNA-mediated downregulation of AMPKα1 and AMPKα2 , as assessed by immunoblot analysis (n = 3). Note that the antibody recognizes both AMPKα1 and AMPKα2.


( c ) Immunoblot detection of ACCα phosphorylation in WT and p53 − / − HCT116 cells subjected to knockdown of AMPKα1 and AMPKα2 ( n = 5 ) ; Co , control .

(c) Immunoblot detection of ACCα phosphorylation in WT and p53−/− HCT116 cells subjected to knockdown of AMPKα1 and AMPKα2 (n = 5); Co, control.


( d ) Quantification of GFP - LC3vac cells ( means ± s . d . , n = 3 separate experiments ) in WT HCT116 and p53 − / − HCT116 treated with PFT - α and / or rapamycin .

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(d) Quantification of GFP-LC3vac cells (means ± s.d., n = 3 separate experiments) in WT HCT116 and p53−/− HCT116 treated with PFT-α and/or rapamycin.


( a ) Cytosolic ATP levels in WT and p53 − / − HCT116 cells in the absence of glucose . Measurements were performed on luciferase - expressing HCT116 , after perfusion of the cells with medium first containing then missing glucose .

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(a) Cytosolic ATP levels in WT and p53−/− HCT116 cells in the absence of glucose. Measurements were performed on luciferase-expressing HCT116, after perfusion of the cells with medium first containing then missing glucose.


( b ) Effect of methylpyruvate ( MetPyr ) on ATP levels in WT p53 − / − HCT116 cells depleted of glucose . ATP levels were measured as in a , in the presence of glucose and 15 min after its withdrawal or replacement by MetPyr .

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(b) Effect of methylpyruvate (MetPyr) on ATP levels in WT p53−/− HCT116 cells depleted of glucose. ATP levels were measured as in a, in the presence of glucose and 15 min after its withdrawal or replacement by MetPyr.


( c ) Effect of autophagy on the ATP levels of WT and p53 − / − HCT116 cells depleted of glucose . Cells were transfected with siRNAs specific for Beclin 1 and AMPKα , or they were treated with rapamycin for 6 h , followed by measurement of ATP levels as in a , before or after glucose withdrawal ( means ± s . d . of triplicates , 3 independent experiments ) .

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(c) Effect of autophagy on the ATP levels of WT and p53−/− HCT116 cells depleted of glucose. Cells were transfected with siRNAs specific for Beclin 1 and AMPKα, or they were treated with rapamycin for 6 h, followed by measurement of ATP levels as in a, before or after glucose withdrawal (means ± s.d. of triplicates, 3 independent experiments).


( d ) Metabolic stress - induced cell death is attenuated in the absence of p53 . HCT116 cells were transfected with control , Atg5 - , Atg10 - , or AMPKα - specific siRNAs and were subjected 48 h later to metabolic stress ( cultured for 48 h in nutrient - free , hypoxic conditions ) and stained with DiOC6 ( 3 ) and PI . The black and white portions of the columns refer to the DiOC6 ( 3 ) low PI − ( dying ) and DiOC6 ( 3 ) low PI + ( dead ) population , respectively .

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(d) Metabolic stress-induced cell death is attenuated in the absence of p53. HCT116 cells were transfected with control, Atg5-, Atg10-, or AMPKα-specific siRNAs and were subjected 48 h later to metabolic stress (cultured for 48 h in nutrient-free, hypoxic conditions) and stained with DiOC6(3) and PI. The black and white portions of the columns refer to the DiOC6(3)low PI− (dying) and DiOC6(3)low PI+(dead) population, respectively.


( e ) Metabolic stress - induced decrease of clonogenic survival was attenuated in the absence of p53 . HCT116 cells subjected to metabolic stress as in d were monitored for clonogenic survival .

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(e) Metabolic stress-induced decrease of clonogenic survival was attenuated in the absence of p53. HCT116 cells subjected to metabolic stress as in d were monitored for clonogenic survival.


( f ) Expression of Beclin 1 in MCF7 cells restores the survival advantage conferred by p53 depletion . MCF7 cells that carry a tetracycline - repressible Beclin 1 expression construct were cultured to avoid Beclin 1 expression or to induce it , transfected with a control siRNA or a p53 - specific siRNA , subjected to metabolic stress , and finally stained with DiOC6 ( 3 ) / PI .

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(f) Expression of Beclin 1 in MCF7 cells restores the survival advantage conferred by p53 depletion. MCF7 cells that carry a tetracycline-repressible Beclin 1 expression construct were cultured to avoid Beclin 1 expression or to induce it, transfected with a control siRNA or a p53-specific siRNA, subjected to metabolic stress, and finally stained with DiOC6(3)/PI.


( g ) Effect of p53 and autophagy on the survival of metabolically stressed MEFs . WT MEFs were transfected with the indicated siRNAs and then subjected to metabolic stress before performing clonogenic assays . Results in d - g are mean ± s . d . of 3 separate experiments ( * P 0 . 05 ) ; Co , control .

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(g) Effect of p53 and autophagy on the survival of metabolically stressed MEFs. WT MEFs were transfected with the indicated siRNAs and then subjected to metabolic stress before performing clonogenic assays. Results in d-g are mean ± s.d. of 3 separate experiments (*P 0.05); Co, control.


( a , b ) PFT - α - mediated stimulation of autophagy in cytoplasts . GFP - LC3 - transfected HeLa cells were enucleated by density - gradient centrifugation after cytochalasin B treatment and cultured on polylysine - coated coverslips . Mixtures of cells and cytoplasts ( arrows , identified by the lack of Hoechst 33342 staining ) were then exposed to three different autophagy inducers : PFT - α , nutrient starvation and rapamycin . Results are means ± s . d . n = 3 experiments ) .

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(a, b) PFT-α-mediated stimulation of autophagy in cytoplasts. GFP-LC3-transfected HeLa cells were enucleated by density-gradient centrifugation after cytochalasin B treatment and cultured on polylysine-coated coverslips. Mixtures of cells and cytoplasts (arrows, identified by the lack of Hoechst 33342 staining) were then exposed to three different autophagy inducers: PFT-α, nutrient starvation and rapamycin. Results are means ± s.d. n = 3 experiments).


( c - e ) Effect of p53 mutants on autophagy in p53 − / − HCT116 cells . Representative micrographs of cells transfected with plasmids expressing WT , nuclear and cytoplasmic p53 are shown in

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(c-e) Effect of p53 mutants on autophagy in p53−/− HCT116 cells.Representative micrographs of cells transfected with plasmids expressing WT, nuclear and cytoplasmic p53 are shown in d