Fig. 6: H. pylori induces R-loop formation that depends on NF-κB and active transcription.

a, b U2OS cells were either infected for 6 h with H. pylori P12 (MOI of 20 or 50), or treated with 100 nM camptothecin (CPT), and were treated or not with doxycycline (−/+ DOX) to induce the expression of a (D210N) mutant version of RNase H1 fused to GFP (RNH1^D210N). Cells were subjected to immunofluorescence staining for 53BP1 and PCNA as well as DAPI. Representative images are shown in RNH1^D210N/GFP foci and 53BP1 foci, alongside scatter dot plots of RNH1^D210N/GFP foci of >1468 and up to 1661 cells per condition in b; the −DOX panel shows lack of background signal in the GFP channel in the absence of RNH1^D210N/GFP expression. Data in b are pooled from three independent experiments. c, d U2OS cells were infected with H. pylori (MOI of 50) or exposed to α- or β-ADP-heptose at 0.5 μM final concentration for 6 h and treated or not with doxycycline (−/+ DOX) to induce the expression of RNH1^D210N/GFP. Cells were subjected to immunofluorescence staining for 53BP1 and PCNA as well as DAPI. Representative images are shown in c of RNH1^D210N/GFP foci and 53BP1 foci, alongside scatter dot plots of RNH1^D210N/GFP foci of >1326 and up to 1565 cells per condition in d. Data in d are pooled from three independent experiments. e Scatter dot plots showing 53BP1 foci of >1000 and up to 3000 cells per condition of the U2OS cells shown in a and c, and their counterparts not treated with doxycycline. f–h U2OS cells were infected with the indicated strains of H. pylori (MOI of 50) and/or exposed to the NF-κB inhibitor BAY 11-7082 (1 μM) or triptolide (100 nM) and treated with doxycycline to induce the expression of RNH1^D210N/GFP. Cells were subjected to immunofluorescence staining for 53BP1 and PCNA as well as DAPI. Representative images are shown in f of RNH1^D210N/GFP foci and 53BP1 foci, alongside scatter dot plots of RNH1^D210N/GFP foci of >405 and up to 868 cells per condition in g, and of 53BP1 foci of >691 and up to 1042 cells per condition in h. Data in g and h are pooled from three independent experiments. Red lines indicate medians throughout. P-values were calculated by one-way ANOVA with Dunn’s multiple comparisons correction. ns, not significant; ***p < 0.005, ****p < 0.0001. Scale bars, 10 μm.