FIGURE 3.
Loss of Pds5A and Pds5B-induced DNA damage, Chk1 phosphorylation, and apoptosis. (A) Immunofluorescence images of Pds5-depleted cells showing the phosphorylation and foci formation of histone H2AX (green) that co-localizes with PCNA (red). DNA was stained with Hoechst 33342 (blue). Merged images are shown on the right. Scale bar: 8 μm. (B) Histogram indicating the percentage of cells with phospho-histone H2AX foci, with the mean ± SEM of at least 100 cells from three independent experiments shown: **p < 0.01; *p < 0.05. P-values were calculated using a two-tailed Students t-test. (C) Comet images of propidium iodide (1 mg/ml)-labeled HeLa cells following a 48 h treatment with 50 nM of either control-si or Pds5A-si. Similar results were obtained with cells depleted of the Pds5B protein. Cells were mixed with low melting point agarose prior to electrophoresis in ice-cold alkali buffer at 30 V, 300 mA for 20 min. Control cells were also X-ray-irradiated (10 Gy). Scale bar: 10 μm. (D) Histogram showing quantitation of data in (C). The DNA damage is expressed as the percentage of DNA in the comet tails. The mean ± SEM of at least 100 cells scored in randomly selected fields from three independent experiments are shown: **p < 0.01; ***p < 0.001; ****p < 0.0001. P values were calculated using a two-tailed Student’s t-test. (E) Western blot analysis of Pds5-depleted cell lysates with antibodies against Pds5A, Pds5B, caspase-3, cleaved caspase-3, PARP, cleaved PARP, and γ-tubulin. Parallel cells were treated with 1 μM of staurosporine for 6 h to induce apoptosis. (F) Western blot detection of p-Chk1S317 in response to DNA stress. Total protein extracts were prepared from synchronized control cells and Pds5A- or Pds5B-depleted cells at the G1/S phase and released for the indicated time points.