Fig. 1
Non-canonical DNA damage response induced by a bacterial genotoxin. a Recombinant typhoid toxin comprising epitope-tagged CdtBFLAG, PltAMyc and PltBHis (asterisk marks E.coli contaminant). MW in kDa left. b Toxin immunoblotted with anti-FLAG (CdtBFLAG, green), -Myc (PltAMyc, red), or -His antibodies (PltBHis, green). MW in kDa left. HT1080s treated with 5 ng/ml wild-type toxin (toxinWT) or catalytically inactive toxinHQ for 2 h before analysis at 24 h as follows: c Representative cell-cycle arrest induced by toxinWT. Propidium iodide (PI) used as DNA marker and fluorochrome (a.u, arbitrary units). Cell-cycle phases indicated. d Representative image of γH2AX (magenta) and 53BP1 (yellow) with outlines of DAPI-stained nuclei. Scale bars 50 μm. e Representative confocal microscopy of γH2AX RING (magenta) and DAPI-stained nuclei (cyan). Side-panels show z-sections. Scale bars 5 μm. f Proportion of cells with γH2AX RINGs and foci. Coloured circles indicate means from technical replicates (2 biological replicates, ~2000 nuclei/variable). Error bars Standard Deviation (SD). g ToxinWT-induced γH2AX RINGs. Incubation times and concentrations indicated. Six technical replicates, ~360 nuclei/variable (one biological replicate). Error bars Standard Error of the Mean (SEM). h Proportion of cells with 53BP1 foci. Coloured circles indicate means from fields of view (3 biological replicates, ~2200 nuclei/variable). Error bars SEM. i Proportion of 53BP1-positive cells with γH2AX RINGs and foci. Coloured circles indicate means from technical replicates (two biological replicates, 450 nuclei/variable). Error bars SEM. j Representative image of γH2AX RINGs (magenta) in infected cells. Salmonella Typhi (green) encoding the toxin (WT) or toxin null mutant (ΔcdtB) at 24 h shown with outlines of DAPI-stained nuclei. Scale bars 50 μm. k Representative image of γH2AX RINGs (magenta) in EcCDT-treated cells shown with outlines of DAPI-stained nuclei. Scale bars 25 μm. In representative images d, j, k arrows indicate DNA damage RINGs (blue) and foci (white). Statistical significance (**** = P < 0.0001, *** = P < 0.0002, ** = P < 0.0021, * = P < 0.0332, ns denotes non-significant = P > = 0.05) calculated using an unpaired two-sided t test (g, i), or relative to corresponding control using one-way ANOVA and Tukey’s multiple comparison test (f, h). Source data provided as a Source Data file
