Fig. 3: Senescence caused by loss of DNA methylation is independent of p53 and p16/Rb.

A γ-H2AX immunofluorescence in HCT116 lines upon hydroxyurea treatment (HU, 2 mM, 4 h) or upon Dox/Auxin treatment. B Immunoblots for total H2AX, γ-H2AX, Chk1, phospho-Chk1 (Ser317), Chk2, phospho-Chk2 (Thr68), and p53 after 8-day Dox/Auxin treatment. HCT116 cells were used as positive controls: Persistent DNA damage response (DDR): Etoposide (10 µM, 24 h), then plain medium for 5 days; Eto-acute: Etoposide, 10 µM, 4 h; HU-acute: hydroxyurea, 2 mM, 4 h. C Scheme of p16 depletion experiments. D Total cell numbers at the indicated days of Dox/Auxin treatment. N = 3 technical replicates. E SA-β-gal staining and quantification of indicated lines. N = 5 fields of view. F RT-qPCR results of p16 (CDKN2A) and selected SASP genes. G Scheme of E6 + E7 expression experiments. H Total cell numbers at the indicated days of Dox/Auxin treatment. N = 3 technical replicates. I SA-β-gal staining and quantification of indicated lines. N = 5 fields of view. J RT-qPCR results of E6, E7, and selected SASP genes. All scale bars are 50 µm. Data of D, E, H, I are presented as mean ± SD and analyzed by two-way ANOVA with Sidak’s multiple comparisons test. Heatmap data of F, J are presented as mean from three technical replicates. In all figures we use the following convention: *p < 0.05, ***p < 0.001, ****p < 0.0001, ns non-significant. Source data are provided as a Source Data file.