Depletion of BuGZ or Bub3 leads to increased R-loop formation and DNA damage. (A) Depletion of BuGZ or Bub3 resulted in increased R-loop formation in HT29 cells. Pladienolide B treatment was used as a positive control. White broken circles outline the nuclei with no or low levels of R-loops. Bar, 5 µm. The quantification of mean fluorescence intensity of the R-loop is shown on the right. Student’s t test: ***, P < 0.001 from three independent experiments. (B and C) Depletion of BuGZ (B) or Bub3 (C) by two different siRNAs in HT29 cells caused increased DNA damage, as judged by the presence of prominent nuclear foci of γ-H2Ax. BuGZ- or Bub3-depleted cells (arrowheads) exhibit more γ-H2Ax foci than cells with insufficient or no depletion (arrows). White broken circles outline the nuclei. Bars, 10 µm. (D–F) Quantification of γ-H2Ax intensities in individual cells (D) and distributions of γ-H2Ax intensities (E and F). Error bars indicate SEM. Student’s t test: ***, P < 0.001. Cell numbers (n) analyzed were from three independent experiments.