Fig 2: CHAF1A recruits RAD18 to stalled replication forks.A Schematic of RAD18-EdU PLA assay. B CHAF1A-knockdown KYSE510 cells were treated with or without 100 J/m2 for 1 h. Biotin was connected to EdU by a Click-iT reaction, and then PLA assay was performed with anti-RAD18 and anti-biotin antibodies. C Statistical diagram of PLA foci for C, n = 100. Data are representative of at least three independent experiments. **P < 0.01; one-way ANOVA. D Schematic of ssDNA/dsDNA pull-down assay. E Biotinylated ssDNA/dsDNA were first bound with streptavidin beads and coated with bacterially produced recombinant CHAF1A, followed by incubating with the indicated concentration of recombinant RAD18. The relative DNA-loaded of RAD18 is calculated by the gray value of RAD18 in F. Data represent means ± SEM from three independent experiments. **P < 0.01; one-way ANOVA. G Biotinylated ssDNA/dsDNA were first bound with streptavidin beads and coated with bacterially produced recombinant wild-type and CHAF1A mutants, followed by incubating with recombinant RAD18, followed by immunoblotting with RAD18 antibody. H Schematic of ssDNA/dsDNA pull-down assay with RPA complex. I Biotinylated ssDNA/dsDNA were first bound with streptavidin beads and coated with bacterially produced recombinant RPA complex (RPA1-His, RPA2-His and RPA3-His), followed by incubation with recombinant CHAF1A and then with recombinant RAD18 at the indicated concentrations. The relative DNA-loaded of RAD18 is calculated by immunoblotting with RAD18 antibody.

Fig 3: Dual inhibition of IMPDH and ATR induces p53-independent replication catastrophe(A) Immunoblot of WaGa cells pre-induced with DOX (1 μg/mL) for 24 h to express p53DD or eGFP followed by treatment with MPA (1 μM) and berzosertib (250 nM) for an additional 24 h. Representative of 3 independent experiments.(B) Immunoblot of MKL-1 p53 KO or control (AAVS1) cells treated with MPA (1 μM) and berzosertib (250 nM) for 3 days. Representative of 3 independent experiments. For panels A and B, the total p53 blot is a reblot of pp53, the total KAP1 blot is a reblot of the pKAP1 blot, the total CHK1 blot is a reblot of pCHK1, and the total RPA32 blot is a reblot of pRPA32.(C) Flow cytometry analysis of chromatin-associated γH2AX and RPA32 in WaGa cells pre-induced and treated as in (A). Gating strategy for each defined population is shown. Images generated in FlowJo. Representative of 3 independent experiments.(D) Quantification of populations from (C). N = 3; mean ± SD.(E) Quantification of chromatin-associated CDC45 from WaGa cells treated as in (A). N = 3; mean ± SD; two-way ordinary ANOVA corrected for multiple comparisons via Tukey post hoc test; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.(F) Quantification of chromatin-associated PCNA from WaGa cells treated as in (A). N = 3; mean ± SD; statistical tests are identical to (E).(G) Quantification of WaGa single cells dual-positive for AV and DAPI prepared as in (A) but treated with inhibitors for 2 days. N = 3; mean ± SD; statistical tests are identical to (E).

Fig 4: NVB S suppresses HGC-27 xenograft tumor growth without causing systemic toxicity. (A) Schematic illustration of the experimental timeline and treatment regimen. (B) Representative photographs of excised tumors from each treatment group at the study endpoint (day 14; n = 7 mouse per group). (C) Tumor growth curves showing volume measurements over time for each group. (D) Quantitative analysis of final tumor weights across treatment groups. (E) Representative immunohistochemical images of tumor sections showing NVB S treatment inhibits tumor progression in mice, with indicators including: (i) H&E staining, (ii) Ki67, (iii) cleaved caspase-3/7, (iv) γ-H2AX and TUNEL, (v) RAD51 staining (scale bar, 50 μm, n = 5 independent experiments). (F) Western blot analysis of γ-H2AX and cleaved caspase-7 in tumor lysates from each group (n = 3 independent experiments). (G) Western blot analysis of p-RPA32 (Ser8) and total RPA32 expression in tumor lysates from each group (n = 3 independent experiments). (H) Western blot analysis of p-CaMKII/CaMKII expression in tumor lysates from each group (n = 3 independent experiments). (Low: NVB S 50 mg/kg; High: 100 mg/kg). All data are presented as mean ± SD. Statistical significance was assessed using One-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001, N.S., not significant