Fig 2: EDC4 interacted with RPA and promotes RPA phosphorylation. a The interaction of EDC4 and RPA1 or RPA2 was analyzed by immunoprecipitation. b The phosphorylation of RPA was analyzed by Western blot

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: Diphthamide affects cellular DNA replication stress through RRM1. (A) Immunoblot analysis of γ-H2AX and p-RPA32 (Thr21) levels in HEK293T WT and DPH4KO cells with RRM1 siRNA knockdown. (B) Immunoblot analysis and quantification of p-RPA32 (Thr21) levels in HEK293T DPH4KO with RRM1-Flag overexpression. (C) Proposed model for diphthamide’s role in affecting mammalian cell DNA replication stress through modulating RRM1 translation. Data with error bars are mean ± s.d. P values are determined by the unpaired Welch’s t test.

Fig 5: 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.