Figure 3.
UV-C induced checkpoint activation in PrimPol−/− cells is partially resolved by inhibition of Chk1 or p38. (A) Chk1 phosphorylation was analyzed by western blotting of whole cell lysates at increasing recovery times (2-24 hrs) after 4 J/m2 UV-C damage. (B) The affect of UCN-01 on cell cycle progression was measured by counting the presence of p-H3 positive mitotic cells. Cells were pre-treated with 100 nM UCN-01 for approximately 2 hrs before irradiation with 0 or 4 J/m2 UV-C, cells were allowed to recover for 0 or 16 hr before the addition of nocadozole to block mitotic exit for 4 hrs. (C) Mitotic segregation was analyzed by staining with DAPI and α-tubulin 16 hrs after cells were damaged with 4 J/m2 in this case cells were pre-treated and then maintained in 100 nM UCN-01 prior to damage. (D) Effect of p38 on cell cycle progression was measured by counting the percentage of p-H3 positive mitotic cells 4 hrs after incubation with nocodazole. Cells were first pre-treated with 2.5 μM SB203580 for 2 hrs followed by irradiation with 4 J/m2 UV-C, and a 16 hr recovery period. (E) The ability of checkpoint inhibitors to release cells from G2 arrest was measured by allowing cells to recover after 0 or 4 J/m2 UV-C for 16 hrs, followed by addition of 100 nM UCN-01 or 2.5 µM SB203580 and 0.5 µM nocodazole for 4 hrs. Mitotic entry was then assessed by p-H3 staining. For all experiments n ≥ 3 independent experiments, error bars represent standard deviation.