Figure 2.

HDAC4‐induced senescence is characterized by DNA damage. (A) Quantitative analysis of the immunofluorescence staining for γH2AX after 2 days of induction of the indicated transgenes in BJ‐TERT cells. Data are expressed as means ± SD, n = 4. Student t‐test: *P < 0.05, **P < 0.01, ***P < 0.005. (B) Quantitative analysis of the immunofluorescence staining for γH2AX after 8 days of induction of the indicated transgenes in BJ‐TERT cells. Data are expressed as means ± SD, n = 4. Student t‐test: **P < 0.01, ***P < 0.005. (C) Immunofluorescence picture of γH2AX positivity after 8 days of induction of the indicated transgenes. Nuclei were stained with Hoechst 33342. Scale bar: 5 μm. (D) Quantitative analysis of nuclear dimension in BJ‐TERT cells following 8 days of induction of the different transgenes. Measures were obtained with imagej. At least 50 nuclei were scored for each condition. The means and the 1^st and 3^rd quartiles are indicated. *P < 0.05, ***P < 0.005. (E) Percentage of γH2AX‐positive cells with the indicated numbers of γH2AX foci in the nuclei of BJ‐TERT cells, after 8 days of expression of the indicated transgenes. (F) Immunoblot analysis using the indicated antibodies in BJ‐TERT cells expressing the different transgenes for 2 or 8 days. Actin was used as loading control. (G) mRNA expression levels of the indicated genes, as measured by qRT‐PCR in BJ/TERT cells expressing the different transgenes following treatment for the indicated days with 4‐OHT. Data are expressed as means ± SD, n = 3. Student t‐test: *P < 0.05, **P < 0.01.