Expression of RNase H1 in BuGZ- or Bub3-depleted HT29 cells reduces γ-H2Ax and phospho-p53 signals. (A) GFP-RNase H1 (RNase), but not GFP, repressed BuGZ depletion-induced DNA damage in HT29 cells. Cells were first transfected by siRNAs for 12 h and then by GFP-RNase H1 or GFP plasmid for 48 h followed by immunofluorescence microscopy analyses. BuGZ-depleted cells with or without GFP fluorescence are indicated by arrowheads or arrows, respectively. The broken lines indicate nuclear boundaries. Bar, 10 µm. (B and C) Quantifications for the mean fluorescence intensity of γ-H2Ax (B) and the intensity distributions (C) of the experiments shown in A. (D) GFP-RNase H1 repressed BuGZ depletion-induced p53 phosphorylation in HT29 cells. Arrowheads and arrows indicate cells with or without GFP-RNase H1, respectively. White broken circles outline the nuclei. (E and F) Quantifications for the mean fluorescence intensity of phospho-p53 (E) and the intensity distributions (F) of the experiments shown in D. (G and H) Quantifications for the mean fluorescence intensity of γ-H2Ax (G) and the intensity distributions (H) in Bub3-depleted and GFP- or GFP-RNase H1–expressing HT29 cells. (I and J) Quantifications for the mean fluorescence intensity of phospho-p53 (I) and the intensity distributions (J) in Bub3-depleted and GFP- or GFP-RNase H1–expressing HT29 cells. Error bars indicate SEM. Student’s t test: *, P < 0.05; **, P < 0.01; ***, P < 0.001. Cell numbers (n) analyzed were from three independent experiments.