FIGURE 3.

Overexpression of H2ABbd induces DNA damage during S phase. A, the experimental strategy to synchronize HeLa cells in G₁/S phase by double thymidine block and induce H2ABbd or H2A expression after the addition of Dox is shown. Cells were released from the thymidine block at 0 h, and samples were taken. B, cells expressing EGFP-H2ABbd and EGFP-H2A were collected at 24 h after release and subjected to MNase analysis. DNA was isolated from cells after MNase treatment for the indicated time (min) and subjected to agarose gel electrophoresis. C, EGFP-H2ABbd-expressing cells were collected at the indicated times after release and subjected to MNase analysis (left). As a control, the data from HeLa cells without induction of H2ABbd are also shown (right). D, cells prepared as in A were collected at the indicated times after thymidine block for FACS analysis. E, total cell extracts were subjected to immunoblotting using the indicated antibodies. F, RPE cells were cultured in medium without FBS for 5 days to synchronize in G₀ (left) or further incubated in the serum-starved condition with Dox for 72 h (right) to express FLAG-HA-tagged H2ABbd. G, RPE cells were cultured in medium without FBS for 5 days and further incubated in the presence of Dox. At 8 or 16 h after the addition of Dox, cells were collected, and total cell extracts were subjected to immunoblotting using antibodies as indicated in the figure.