Fig. 5.

Histone H2AX is required for senescence-associated phenotypes. (A) HCA2 cells were infected with lentiviruses expressing short hairpin RNAs directed against eGFP (control, shGFP) or H2AX (shH2AX-A to shH2AX-D). Forty eight hours later, cells were selected for 4 days and allowed to recover for 3 days. Whole-cell lysates were analyzed by western blotting for the indicated proteins. (B) HCA2 cells were infected with lentiviruses expressing shGFP (control) or shH2AX-A–shH2AX-D, as in A. Infected cells were irradiated with 8 Gy. Two days later, they were stained for the indicated DDR proteins. (C) HCA2 cells were infected with lentiviruses expressing shGFP or shH2AX-A–shH2AX-D. Infected cells were irradiated with 10 Gy. 7 days later, BrdU was added for 24 hours. Top panels: cells were fixed and stained for H2AX (grayscale). Lower panels: cells were fixed and stained for DNA synthesis (BrdU incorporation, red) and DNA (DAPI, blue). (D) Cells in A were treated as in B and analyzed for DNA synthesis (BrdU positive). Shown are the means ± s.d. from three or more independent measurements. (E) HCA2 cells were infected as in A and either untreated or irradiated with 10 Gy. After 2 days, conditioned media were collected over an interval of 24 hours from untreated cells (‘Controls’) and irradiated cells 2 days after irradiation (2–3 days) and 9 days after irradiation (9–10 days). Conditioned media were assessed for IL-6 by ELISA. IL-6 secretion is reported as 10^–6 pg per cell per day. Shown are the means ± s.d. from three or more independent measurements.