Fig. 5

ISP I triggers DNA damage, R-loop formation, and alters the localization of R-loops. A and B Immunofluorescence staining assay (A) and western blots (B) show γH2AX expression in LN229 and U251 cells treated with ISP I at indicated concentrations for 6 h. The number of γH2AX foci in cell nucleus was identified and quantified. Expression of GAPDH serves as an internal control. C and D Immunofluorescence staining assay shows R-loop formation in U251 cells. U251 cells were transfected with V5-tagged catalytically dead RNaseH1 for recognition of R-loops, and then treated with ISP I at indicated concentrations for 30 min. U251 cells were co-stained with antibodies recognizing R-loop (V5, red), DNA replication (EdU, green), nucleolus (nucleolin, blue) or DNA damage (γH2AX, blue). Cell nuclei were counterstained with DAPI (purple). Representative immunofluorescence staining images were shown in (C). Quantitative comparations of ISP I’s effect on R-loop formation are shown in (D). E Representative images show colocalization of R-loops (red) with γH2AX (blue). F Quantitative comparations of ISP I’s effect on DNA damage show an increase in γH2AX foci formation in U251 cells. G The number of R-loops on DNA damage sites were identified and counted by the colocalization of R-loops with γH2AX. H Representative image shows no colocalization of R-loops (red) with EdU (green). I Quantitative comparisons of ISP I’s effect on EdU incorporation show a reduction in EdU incorporation in U251 cells. J The number of R-loops on DNA replication sites were identified and quantified by the colocalization of R-loops with EdU. All data are shown as mean ± SEM. P value: *p < 0.05; **p < 0.01; ***p < 0.001