Figure 4. Proliferation and DNA damage response are impaired in DM1-derived fibroblasts.

(A) Growth of DM1-derived fibroblasts (n = 6) compared with control fibroblasts (n = 5), analyzed as the number of cells at day 1, 3, and 5. (B) Representative image and quantification of pH3⁺ (Ser10⁺) cells in controls (n = 6) and DM1 fibroblasts (n = 6). Scale bar: 100 μm. (C) Representative image and quantification of EdU⁺ cells in controls (n = 2) and DM1 fibroblasts (n = 4). Scale bar: 50 μm. (D) Quantification of Ki67⁺ cells in controls and DM1 myoblasts (n = 2) at early passage (n ≤ 5). (E) Representative image and quantification of γ-H2AX⁺ cells and phospho-ATM detection in controls and DM1 fibroblasts in the absence or presence of doxorubicin (n ≥ 3). Scale bar: 50 μm. (F) Quantification of γ-H2AX⁺ cells in indicated conditions. (G) Representative immunoblot of p53 detection in controls and DM1 fibroblasts in the absence or presence of doxorubicin (n = 3). (H and I) Representative immunoblot and quantification of p16^INK4A, p21^CIP1, and p27^KIP1 protein levels in DM1 compared with control fibroblasts (each point represents an independent sample). (J) mRNA levels of p16^INK4A, p14^ARF, p27^KIP1, and p21^CIP1 in fibroblasts (n = 4 for controls and n = 6 for DM1). (K) mRNA levels of BMI1 in the same fibroblasts as J. (L) mRNA levels of indicated genes in early passage myoblasts (n ≤ 7) derived from patients with DM1 and controls (each point represents an independent experiment). (M) Measurement of mRNA levels of indicated genes in tibialis anterior muscle of 2-month-old DMSXL and control mice (n ≤ 6). (N) p16^INK4A, p14^ARF, p21^CIP1, p27^KIP1, and BMI1 mRNA levels in PBMCs from patients with DM1 (n ≥ 56) and controls (n ≥ 22). P values were calculated using the Student’s t test. ^≠P < 0.1, *P < 0.05, **P < 0.01, ***P < 0.001.