Figure 6.

PPM1G Depletion Results in Accumulation of p53BP1 Foci and p53-Independent Cell-Cycle Arrest

(A–C) AG06173 cells were either left untreated (Control) or treated with Lipofectamine RNAiMAX transfection reagent in the presence of GFP or PPM1G siRNA (200 pmol) for 48 hr. In (A) and (B), cells were collected by trypsinisation and subjected to FACS analysis. Cell-cycle profiles and quantification of cell cycle distribution are shown, respectively. In (C) cells were pelleted by centrifugation, whole-cell extracts were prepared and analyzed by western blotting.

(D–F) AG06173 cells were grown on coverslips for 24 hr to 30%–50% confluency, left untreated (Control) or treated in the presence of GFP or PPM1G specific siRNA as described above and then immunostained with p53BP1 antibodies followed by Alexa Fluor 488 anti-rabbit IgG. Detection of p53BP1 foci formation was performed, the obtained data were analyzed and presented as the number of foci per cell normalized to Control (D) and as the percentage of cells containing three or more foci per nucleus (E). Statistical data are presented as a mean ± SD of three to five independent biological experiments. An example of p53BP1 foci formation in PPM1G-depleted AG06173 cells is shown (F).