Fig 2: RPA knockdown reversed the inhibitory effect of EDC4 on cisplatin-induced DNA damage. a The level of EDC4, RPA1 and RPA2 in cells with EDC4 overexpression (or RPA knockdown) analyzed by Western blot. b The cell growth of HeLa (with EDC4 overexpression with/or RPA knockdown) evaluated by colony formation assay. c The DNA damage of HeLa (with EDC4 knockdown) analyzed by γH2AX immunofluorescent staining. **p < 0.01 vs. Vector; ##p < 0.01 vs. EDC4 + siNC

Fig 3: CBX3 is recruited to stalled replication forks in an RPA2-dependent manner.(A) FLAG-tagged CBX3 was immunoprecipitated from 293T cells and subjected to MS analysis to identify CBX3 interacting proteins. (B) The list of top hits proteins from CBX3 MS. (C) Western blot (WB) analysis of whole-cell lysates (WCL) and anti-CBX3 immunoprecipitations (IPs) derived from PC-3 cells. (D) WB analysis of WCL and anti-RPA2 IPs derived from PC-3 cells. (E) Colocalization of CBX3 with RPA2 foci after noted drug treatment or transfected with indicated small interfering RNA (siRNA) in U2OS cells. Scale bar, 10 μm. (F) Quantitation of average CBX3 foci number in each cell. Data were presented as means ± SD of more than 200 cells from three biological replicates. n.s., not significant. Two-tailed unpaired Student’s t test, ***P < 0.001. (G) Replication fork-bound proteins were isolated by the iPOND assay with control and RPA2 knockdown U2OS cells. (H) Schematic diagram depicting a set of GST-RPA2 recombinant protein constructs. (I) WB analysis of Flag-CBX3 proteins in U2OS cells pulled down by GST or GST-RPA2 recombinant proteins. (J) Schematic diagram depicting a set of GST-CBX3 recombinant protein constructs. (K) WB of His-RPA2 proteins in U2OS cells pulled down by GST or GST-CBX3 recombinant proteins. (L and M) U2OS cells transfected with Flag-CBX3 proteins were subjected to immunofluorescence staining (IFC) after HU treatment. Representative images were shown in (L) and quantitative data are shown in (M). 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. IgG, immunoglobulin.

Fig 4: Working model illustrating how CBX3 regulates genome stability by trapping RPA2 at stalled replication forks and inducing replication stress and enabling synthetic lethality to PARP inhibition.Under normal conditions, phosphorylation of CBX3 serine-95 by CK2 kinase augments CDH1-mediated CBX3 degradation and RPA2 dynamics at stalled replication forks, thereby promoting replication restart. However, prostate cancer–associated amplification or CK2 inhibitor–induced CBX3 high expression inhibits RFWD3 binding with RPA2, which consequently traps RPA2 at stalled replication forks, resulting in replication stress and enabling synthetic lethality to PARP inhibition.

Fig 5: 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.