Figure 3.

S phase-specificity of DNA damage responses by Ni(II). Experiments were done with H460 cells, except for panel F using primary WI38 cells. H460 cells were synchronized in G1 phase by incubations with the CDK4/6 inhibitor PD-0332991 (0.5 μM, 16 h), in G2 phase by treatments with the CDK1 inhibitor RO3360 (10 μM, 16 h) and in S phase by releasing from the G1 arrest and addition of Ni(II) at 3 h post-release. All Ni(II) treatments of H460 cells were for 6 h. A, FACS profiles of propidium iodide-stained control and PD0332991-treated cells (top panel) or control and RO3360-treated cells (bottom panel). B, progression of H460 cells through cell cycle after their release from G1 arrest. C, activation of p53 and CHK1-S317 phosphorylation by Ni(II) in different cell cycle phases. D, scoring of p53-S15 phosphorylation in EdU-positive (S-phase) and EdU-negative H460 cells treated with 0 or 400 μM Ni(II) for 6 h (sh-ns: nonspecific shRNA, sh-p53: p53-targeting shRNA). Data are means ± SD, n = 3. Insert: immunoblot demonstrating p53 knockdown by shRNA in control and Ni(II)-treated cells. E, hypersensitivity of S-phase cells to Ni(II) cytotoxicity. Asynchronous and S-phase-synchronized cells were treated with Ni(II) for 6 h and cell viability measurements were taken 48 h later. Data are means ± SD, n = 3, ∗∗p < 0.01. F, proliferation-dependent formation of γ-H2AX by Ni(II) in primary WI38 cells. Proliferating cells and cells arrested in G1 (24 h with 0.5 μM PD-0332991) or G2 phases (10 μM RO3360, 24 h) were treated for 24 h with Ni(II). G, time-dependent phosphorylation of p53 and CHK1 by 600 μM Ni(II) in H460 cells.