Figure 4. [Ru(dppz)2(PIP)]2+ induces Chk1 checkpoint kinase activation and G1-S cell-cycle arrest.
(a,b) Whole-cell extracts of HeLa cells treated with 1 (40 μM) or mitoxantrone (MX, 2 μM) for 3, 8 or 24 h (a) or a concentration gradient of 1 or 2 (24 h incubation time, b) were immunoblotted for activated (phosphorylated, p) DNA damage response (DDR) signalling proteins pChk1 (Ser345), pBRCA1 (Ser1524), pChk2 (Thr68) and γH2AX (pH2AX at Ser319), as indicated. β-actin was used as a loading control and cisplatin employed as a positive control for DDR activation. Concentration ranges for 1 and 2 were centred on IC50 values, Table 1. See SI for densitometry. (c) Cell-cycle distributions of HeLa cells incubated with DMSO (0.2%), 1 or 2 at the stated concentrations and time. DNA content was quantified using propidium iodide (PI) and analysed by flow cytometry. Cisplatin (10 μM) and MX (2 μM) treatment were included for comparison. Data mean of three independent experiments +/− SD.