Fig. 4
Typhoid toxin exhausts RPA by oversupply of ssDNA substrate. a Representative image of toxin-induced RPA exhaustion. HT1080 cells intoxicated with toxinWT or toxinHQ for 2 h before polymerisation of double-stranded BrdU-labelled DNA from sites of exposed ssDNA template at 24 h (ssDNA, magenta). ssDNA-bound RPA32 pT21 (pRPA, cyan) shown with BrdU shown in grayscale for clarity (ssDNA foci). Insets of toxinWT magnify ssDNA foci unprotected by pRPA (blue arrow) or partially protected by pRPA indicated by white co-localisation staining (white arrow). Insets of toxinHQ show RPA-bound ssDNA. Scale bars 10 μm. b Proportion of BrdU-labelled ssDNA foci per nucleus in intoxicated HT1080 cells. Aphidicolin (APH) was used as positive control for ssDNA induction and toxinHQ used as a negative control. Coloured circles indicate means from fields of view. Two biological replicates (160 nuclei per variable). Error bars SEM. c Proportion of serum-starved cells with RPA32 pT21 foci (pRPA). Coloured circles indicate means from technical replicates (two biological replicates, 900 nuclei/variable). Error bars SEM. d Proportion of cells with RPA32 pT21 foci (pRPA) in presence of serum. Coloured circles indicate means from technical replicates (2 biological replicates, 900 nuclei/variable). Error bars SD. e Representative image of RPA32 pT21 (pRPA, grayscale) with γH2AX (magenta) RINGs (blue arrows) in cells treated with APH as indicated. Scale bars 50 μm. f Proportion of γH2AX RINGs in APH-treated cells ± toxinWT as indicated. Coloured circles indicate means from fields of view (two biological replicates, 600 nuclei/variable). Error bars SEM. g Mean RPA pT21 (pRPA) foci intensity in APH-treated cells ± toxinWT as indicated. Coloured circles indicate means from fields of view (two biological replicates, 600 nuclei/variable). Error bars SEM. A.U, arbitrary units. In representative images (a, e) outlines of DAPI-stained nuclei are shown. Statistical significance (**** = P < 0.0001, *** = P < 0.0002, ** = P < 0.0021, * = P < 0.0332, ns = P > = 0.05) was calculated relative to toxinHQ using one-way ANOVA and a Dunnett’s multiple comparison test (b), or relative to untreated via one-way ANOVA and a Tukey’s multiple comparison test (c, d, f, g). Source data provided as a Source Data file
