Figure 5.

BLM response to DNA damage depends on the G2 delay. WI-38 (a–e), AT-2SF, or WI38-E6 (f) cells were treated with the indicated agents while quiescent (a) or proliferating (b–f). Protein lysates were prepared from untreated cells (0 h) or at the indicated times after treatment (h), and analyzed for BLM and α-tubulin (Tubulin; control) by Western blotting. (a) Quiescent cells were X-irradiated (5 Gy) and immediately stimulated with serum. Parallel cultures were pulsed for 24 h with [³H]thymidine to determine the percentage of cells that synthesized DNA (% LN). (b) Proliferating cells were X-irradiated (5 Gy) and immediately given medium containing 5 mM caffeine. Parallel cultures were analyzed for mitotic figures (Mitotic index; 1,000 nuclei/point). The mitotic index of the unirradiated culture was given a value of 1. (c) Cells were given bleomycin (10 μg/ml) for 1 h in serum-containing medium. (d) Cells were given etoposide (10 μM) for 1 h in serum-containing medium. (e) Cells were irradiated with UVC (1.6 J/m²/s) in PBS and returned to serum-containing medium. (f) Proliferating AT-2SF or WI38-E6 cells were X-irradiated (5 Gy), protein lysates were prepared, and BLM protein level was normalized to α-tubulin.