Fig 1: CBX3 antagonizes RFWD3-mediated RPA2 polyubiquitination and promotes RPA2 retention at stalled replication forks.(A and B) U2OS cells infected with lentivirus expressing control or shCBX3 were subjected to IFC after HU treatment. Representative images were shown in (A) and quantitative data are shown in (B). Scale bar, 10 μm. Data were shown as the mean ± SD from three biological replicates, two-tailed unpaired Student's t test, ***P < 0.001. (C) Replication fork-bound proteins were isolated by the iPOND assay with control and CBX3 knockdown U2OS cells. (D) WB analysis of WCL and anti-His IPs derived from PC-3 cells transfected with indicated plasmids followed by HU treatment. (E) WB analysis of WCL and anti-RPA2 IPs derived from PC-3 control and CBX3 knockdown cells transfected with indicated plasmids followed by HU treatment. (F) WB analysis of WCL and anti-His IPs derived from PC-3 cells transfected with indicated plasmids followed by HU treatment. Both poly-ubiquitinated and primary-ubiquitinated RPA2 (asterisk) were observed. (G) WB analysis of WCL and anti-RPA2 IPs derived from PC-3 control and CBX3 knockdown cells transfected with indicated plasmids followed by HU treatment. Both poly-ubiquitinated and primary-ubiquitinated RPA2 (asterisk) were observed. (H and I) U2OS control or CBX3 knockdown cells were reconstituted with WT or V22M mutant CBX3 and subjected to IFC after HU treatment. Representative images were shown in (H) and quantitative data were shown in (I). Scale bar, 10 μm. Data were shown as the means ± SD from three biological replicates, two-tailed unpaired Student's t test, ***P < 0.001. (J) WB analysis of WCL and chromatin binding proteins derived from PC-3 control and CBX3 knockdown cells transfected with indicated plasmids followed by HU treatment.
Fig 2: 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
Fig 3: Both trabectedin and lurbinectedin induce the DNA damage responseA. HeLa cells were mock-treated (UT) or incubated for 1 hour with trabectedin (20 nM) followed by 0, 2, 4, 6 hours post-incubation in drug-free media as indicated. Total protein extracts were prepared and analyzed by immunolabeling with antibodies directed against Ser317-phosphorylated Chk1, Thr68-phosphorylated Chk2, Thr21-phosphorylated RPA32 and Ser139-phosphorylated H2AX. Total Chk1, Chk2, RPA32 and H2AX were used as loading controls. B. HeLa cells were either untreated (UT) or incubated for 1 hour with 20 nM trabectedin (T) or lurbinectedin (L) followed by 6 hours post-incubation in drug-free media. Total protein extracts were prepared and analyzed by immunolabeling with antibodies directed against Ser317-phosphorylated Chk1, Thr68-phosphorylated Chk2 and RPA32. Total Chk1 and Chk2 were used as loading controls. On each panel, arrows indicate the main phosphorylated forms of RPA32.
Fig 4: Diphthamide deficiency contributes to cellular DNA replication stress. (A) Immunoblot analysis and quantification of γ-H2AX levels in HEK293T WT and DPH4KO cells under normal growth conditions. (B) Immunofluorescence staining of γ-H2AX and quantification of γ-H2AX total area in the nuclei of HEK293T WT and DPH4KO cells under normal growth conditions. (C) Immunoblot analysis and quantification of p-RPA32 (Thr21) levels in HEK293T WT and DPH4KO cells under normal growth conditions. (D) Immunofluorescence analysis showing percentage of p-RPA32 (Thr21) nuclei in HEK293T WT and DPH4KO cells. (E) Cartoon illustration of DNA replication fork stalling and immunofluorescence analysis of p-RPA32 (Thr21) foci in HEK293T WT and DPH4KO cells. Representative data from three biologically independent experiments. Data with error bars are mean ± s.d. P values are determined by the unpaired Welch’s t test. Scale bars: 20 μm.
Fig 5: RINGs mark RPA exhaustion induced by the typhoid toxin. a Representative immunoblot of HT1080 cells intoxicated with toxinWT or toxinHQ (txHQ) for 2 h before analysis with indicated antibodies at time-points marked in hours (hr). MW in kDa left. b Proportion of intoxicated cells with γH2AX RINGs or γH2AX foci containing RPA pT21 foci (pRPA, grayscale). γH2AX labelled in magenta. 2 biological replicates (1500 nuclei per variable). Error bars SD. c Representative image of RPA32 pT21 (pRPA) with γH2AX RINGs (blue arrow) or foci (white arrow). Scale bars 50 μm. d Representative immunoblot of RPA knockdown (siRPA) cells shown with transfection (siGAPDH) and gel loading (tubulin) controls. In all, 25 nM siRNA for 48 h. MW in kDa left. e Cell-cycle phases in RPA knockdown or siGAPDH control (ctrl) cells. Coloured symbols indicate means from 3 biological replicate (60,000 cells per variable). Error bars SD. f Representative image of γH2AX RINGs (white arrow) in RPA knockdown cells. Scale bars 25 μm. g Proportion of γH2AX RINGs in RPA knockdown cells ± toxinWT. Non-transfected control (nt). Circles indicate means from fields of view in two biological replicates (122 nuclei per variable). Error bars SEM. h Representative immunoblot of super-RPA cells ± toxinWT analysed with indicated antibodies. Hyper-phosphorylated RPA32 (red arrow), non-phosphorylated RPA32 (blue arrow). MW in kDa left. i Proportion of γH2AX RINGs in super-RPA cells ± toxinWT. Two biological replicates (2000 nuclei per variable). Error bars SEM. j Representative image of γH2AX in U2OS control cells (ctrl) or super RPA U2OS cells stably expressing recombinant RPA complex intoxicated with 5 ng/ml toxinWT. Scale bars 5 μm. In representative images c, f, g 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 between indicated experiments using an unpaired two-sided t test (b), or calculated relative to indicated controls using one-way ANOVA (g, i) or two-way ANOVA (e) together with a Tukey’s multiple comparison test. Source data are provided as a Source Data file
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