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. 2008 Aug 29;9(10):1019–1026. doi: 10.1038/embor.2008.162

Figure 2.

Figure 2

PP4 reduces γH2AX independently of a role in DNA repair in vivo. (A,B) U2OS cells were transfected with siRNAs against PP4C, PP2ACα/β (A), PP4R2 (B) or a non-targeting control sequence. Cells were irradiated with 10 Gy and collected at the indicated times after IR for immunoblotting with the indicated antibodies. Signal intensity was analysed using Image-J (http://rsb.info.nih.gov/ij/). Signal intensity of γH2AX is normalized against that of histone H3. (C) Cells were irradiated with 50 Gy and collected at the indicated times after IR for neutral comet assays. A representative image of the nuclei is presented. (D) Quantification of the comet tail moments of the experiment in (C). Tail moments for each condition were calculated on a minimum of 75 cells for each data point, normalized against the average comet tail moment of the mock-irradiated control, and shown as a box-and-whisker plot. Outliers are indicated as dots. The ordinate is a square-root scale. Data were statistically analysed using the Wilcoxon test. P-values are adjusted for several comparisons with the Bonferroni method. *P<0.05; **P<0.01; ***P<0.0001; NS, no significance. (E) U2OS cells transfected with siRNAs against either PP4C or a control sequence were irradiated with 10 Gy. At 1 h post-IR, the ATM (KU55933) and DNA-PK (KU57788) inhibitors were added to the tissue culture medium (t=0). Samples were then collected at the indicated time points and processed for immunoblotting with the indicated antibodies. Signal intensity was analysed using Image-J. Signal intensity of γH2AX is normalized against that of histone H3. ATM, ataxia telangiectasia mutated; DNA-PK, DNA-dependent protein kinase; H3, histone H3; IR, irradiation; siRNA, small interfering RNA.