Fig. 2: TOP1 poisons increase nuclear R-loops and induce R-loop-mediated γH2AX foci in HeLa cells.

Cells were treated for indicated times with CPT (a) or LMP776 (b) and nuclear R-loops were detected by co-staining with DAPI (blue), anti-hybrid S9.6 (green) and anti-nucleolin (red) antibodies. Quantitative plots on the right represent nucleoplasmic S9.6 fluorescence normalised over untreated cells. Dots show single-cell values and red bars are means for each sample. Asterisks indicate statistical significance in comparison with untreated cells by the Kolmogorov–Smirnov test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. c Immunofluorescence analysis of U2OS_RH cells to detect γH2AX foci (Ser139 phosphorylation, green) and RNaseH1 expression (red) after the indicated times. d, e nuclear γH2AX levels in U2OS_RH cells treated with CPT or LMP776, respectively. Cells exposed to doxycycline were split into three groups with low, intermediate or high RNaseH1 expression levels, respectively (low, intermediate and high). Asterisks indicate statistical significance in comparison with untreated cells by the Mann–Whitney test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Scale bars: 10 μm.