Fig 2: DMD hiPSC-CMs exhibit p53 upregulation(A–E) Representative micrographs of (A) p53 activation by immunoblotting (n = 3 independent experiments), (B) DNA damage 53BP1 foci by immunofluorescence (n = 6 independent experiments), and (C) p21 (n = 6 independent experiments) in cardiac troponin T+ hiPSC-CMs are shown and (D and E) quantified, respectively.(F) Reduced expression levels of PGC-1α, master regulator of mitochondrial biogenesis, determined by qRT-PCR (n = 6 independent experiments).(G and H) (G) Mitochondria amount (n = 6 independent experiments) and (H) mitochondrial copy number (n = 6 independent experiments) in hiPSC-CMs were assessed by MitoTracker Green and qRT-PCR using mitochondrial gene (Nd2) to nuclear DNA (Nrf1) primers, respectively. Data represent mean ± SEM. Student's t was test used for statistical analysis.(I) Telomere loss prevented when contraction of DMD hiPSC-CMs was inhibited between days 20 and 30 using blebbistatin was quantified (n = 3 independent experiments, 35–1,834 cells analyzed). Data represent mean ± SEM. Student's t test was used for statistical analysis.

Fig 3: 53BP1 Is a Robust DNA Damage Marker for Foci Detection(A) Representative time response of background corrected γH2AX (left) and 53BP1 (right) RIFs per nucleus in MCF10A exposed to 1 Gy of 1 GeV/n 56Fe ions.(B) Representative image of two MCF10A nuclei at 30 min post-56Fe irradiation in overlay, DAPI, γH2AX, and 53BP1 channels, illustrating the co-localization of both RIF markers. Scale bar: 5 µm.(C) Representative images of PBMC nuclei (DAPI) and 53BP1+ foci (fluorescein isothiocyanate [FITC]) at 4 and 24 h after 1 Gy of gamma rays or 3 particles/100 µm2 (0.82 Gy) of 600 MeV/n 56Fe ions and nonirradiated controls. Scale bar: 50 µm.

Fig 4: Sol8 Gne KO cells cultured in stress conditions. (A), confluency curves determined by Incucyte (in duplicates; each point depicts the average of 9 fields). (B). Morphology of the cells by phase contrast microscopy. (C), determination of cell size at day 3 (n > 300 cells). (D), staining of the cells with anti alpha actinin Ab. (E), confluency curves of Sol8 Gne KO cells after AAVGNE infection. (F), staining of the infected cells with anti GNE Ab. (G), Immunostaining of Sol8 Gne KO and WT cells for p53BP1; (H), Image -Pro Plus 7 analysis of area and intensity of γ-H2AX staining (relative units). (I), FACS analysis of γ-H2AX stained cells (MFI units) (n = 3 replicates).

Fig 5: KDM2A regulates H3K36me2 at ALT telomeres.a Western blot analysis of ATRX protein expression in whole-cell lysates prepared from the indicated non-ALT and ALT-dependent (ALT+) cells. b Western blot analysis of ATRX expression in whole-cell lysates prepared from control or ATRX-depleted IMR90-T cells. The ATRX-depleted lines (dATRX clone#1–7) were established from clonally isolated IMR90-T cells transduced with ATRX-targeted sgRNA. The lysates from U2OS and ALT#1 cells were included as negative controls of ATRX expression. c Representative immuno-FISH images of 53BP1 and telomeres in ALT#1, IMR90-T (T#1), or ATRX-depleted IMR90-T (dATRX#1 and dATRX#2) cells. Scale bar, 10 µm. d Percentages of cells containing =4 53BP1-associated telomere dysfunction-induced foci (TIFs). Data were expressed as mean ± s.e.m. of three independent experiments; two-tailed unpaired t-test. e Competition-based proliferation assay of KDM2A-targeted sgK#1 (linked with GFP expression) in the indicated cell lines. Data were expressed as mean ± s.e.m. of three independent experiments. f Western blot analysis of Flag and ACTB (loading control) using whole-cell lysates prepared from control or Flag-tagged wild-type KDM2A-transduced HeLa, ALT#1, or Saos2 cells. g, h Telomere dot-blot analysis (g) and quantification (h) of anti-Flag or IgG chromatin immunoprecipitation (ChIP) in the indicated cell lines. The IgG ChIP was included as a control for non-specific signals. The input and ChIP DNAs processed against the indicated antibodies were assayed by dot-blotting and hybridized with a 32P-labeled TelG probe. The relative enrichment was calculated after the normalization of ChIP DNA signals to the respective input DNA signals. Data were expressed as mean ± s.e.m. of three independent experiments; two-tailed paired t-test. i, j Telomere dot-blot analysis (i) and quantification (j) of anti-H3K36me2, anti-H3, or IgG ChIP in sgCtrl or sgK#1-transduced Saos2 cells. The relative enrichment was calculated after normalization to the respective ChIP DNA signals of sgCtrl-transduced samples. Data were expressed as mean ± s.e.m. of three independent experiments; two-tailed paired t-test. k Western blot analysis of KDM2A, H3K36me2, and H3 in cell lysates prepared from sgCtrl or sgK#1-transduced Saos2 cells. Source data are provided as a Source Data file.