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. Author manuscript; available in PMC: 2008 Feb 12.

Published in final edited form as: Clin Cancer Res. 2006 Dec 1;12(23):6884–6893. doi: 10.1158/1078-0432.CCR-06-0410

Fig. 5 — TOPK inhibits As³⁺-induced apoptosis through phosphorylation of histone H2AX at Ser 139. A, effect of TOPK deficiency on H2AX phosphorylation. Control siRNA and TOPK siRNA cells were cultured and treated with 2.5 μM As³⁺ for 24 h. Phosphorylation of histone H2AX at Ser139 and TOPK at Thr9 was visualized by Western blotting using phospho-specific antibodies. Total TOPK, H2AX and β-actin were used as internal controls for confirmation of TOPK deficiency and equal protein loading. B, As³⁺ induced phosphorylation of H2AX in H2AX^+/+ cells, but had no effect on phosphorylation of TOPK in H2AX^+/+ or H2AX^−/− cells. Total TOPK, H2AX and histone H3 were used as internal controls for confirmation of H2AX deficiency and equal protein loading. C, flow cytometry analysis of apoptosis in H2AX^+/+ and H2AX^−/− cells and D, flow cytometry analysis of apoptosis in control siRNA or TOPK siRNA cells. Cells were incubated with annexin V-conjugated FITC after 24 h treatment with 2.5 μM As³⁺. Stained cells were analyzed by flow cytometry. Lower-right panel indicates the percentage of early apoptotic cells (annexin V-stained positive cells). These data are representative of at least four independent experiments.

Fig. 5 — TOPK inhibits As³⁺-induced apoptosis through phosphorylation of histone H2AX at Ser 139. A, effect of TOPK deficiency on H2AX phosphorylation. Control siRNA and TOPK siRNA cells were cultured and treated with 2.5 μM As³⁺ for 24 h. Phosphorylation of histone H2AX at Ser139 and TOPK at Thr9 was visualized by Western blotting using phospho-specific antibodies. Total TOPK, H2AX and β-actin were used as internal controls for confirmation of TOPK deficiency and equal protein loading. B, As³⁺ induced phosphorylation of H2AX in H2AX^+/+ cells, but had no effect on phosphorylation of TOPK in H2AX^+/+ or H2AX^−/− cells. Total TOPK, H2AX and histone H3 were used as internal controls for confirmation of H2AX deficiency and equal protein loading. C, flow cytometry analysis of apoptosis in H2AX^+/+ and H2AX^−/− cells and D, flow cytometry analysis of apoptosis in control siRNA or TOPK siRNA cells. Cells were incubated with annexin V-conjugated FITC after 24 h treatment with 2.5 μM As³⁺. Stained cells were analyzed by flow cytometry. Lower-right panel indicates the percentage of early apoptotic cells (annexin V-stained positive cells). These data are representative of at least four independent experiments.