Fig 2: Downregulation of TOPBP1 sensitizes animal models to olaparib. A Schematic diagram of the mouse model. B Tumor volumes were measured in implanted mouse models of Patu8988 nc and Topbp1-knockdown following 21 days of olaparib treatment. Results show that Topbp1 knockdown increased drug sensitivity to olaparib in mice injected with Patu8988 cells. C Hematoxylin and eosin (HE) and Ki67 immunochemical staining were performed on tumor tissues from Patu8988 nc and Topbp1-knockdown mice treated with either vehicle or olaparib. The positive Ki67 staining was significantly reduced in Topbp1-knockdown tumors treated with olaparib compared to WT tumors treated with olaparib

Fig 3: The influential role of TOPBP1, ATR, ATM, and DDR pathways in the inhibitory effect of olaparib on pancreatic cell proliferation. A Heat map of the differential TOPBP1, ATR, and ATM gene expression in four groups based on BRCA1 mutation status and PARP sensitivity: NOBRinse (BRCA1 non-mutation and PARP insensitivity), BRinse (BRCA1 mutation and PARP insensitivity), NOBRse (BRCA1 non-mutation and PARP sensitivity), and BRse (BRCA1 mutation and PARP sensitivity). B Volcano plots illustrating the increased expression of TOPBP1 and ATR in comparison to the BRCA1 non-mutation and PARP-sensitivity groups, as well as the BRCA1 non-mutation and PARP-insensitivity groups. C Determination of PDAC cell line sensitivity to olaparib using the CCK8 assay. D Heatmap showing differences in signaling pathway enrichment alterations induced by olaparib among BXPC3 and Patu8988 cells treated with olaparib based on Gene Ontology (GO) Biological Processes (BP). E Heatmap showing differential gene expression in BXPC3 and Patu8988 cells under olaparib treatment. F Differential changes in TOPBP1 and other DDR genes analyzed by western blot and ImageJ in PDAC cell lines treated with olaparib and expressing different levels of TOPBP1

Fig 4: Pathway changes in PDAC cell lines with different TOPBP1 levels after olaparib and TOPBP1-knockdown treatment. A–C IC50 values determined by the CCK8 assay showing that TOPBP1 knockdown impairs the drug sensitivity of Patu8988 and Panc1 cells to olaparib compared to the control treatment; this is not apparent in BXPC3 cells. D Differential changes in DDR genes analyzed by western blot and ImageJ in Patu8988 and BXPC3 cells after TOPBP1 knockdown and treatment with 5 μM olaparib. E. Effect of olaparib on apoptosis in Patu8988 nc or shTOPBP1 cells assessed by FACS; the proportion of Annevin V/7-AAD cells is shown. PDAC cells were treated with 5 μM olaparib for 48 h. Significant differences were observed between TOPBP1-knockdown and control cells after olaparib treatment. F Effect of olaparib on cell cycle arrest analyzed by FACS; the proportion of cells positive for FL2-H-PI staining is shown. Patu8988 WT or TOPBP1-knockdown cells were treated with 5 μM olaparib for 48 h; combined olaparib and TOPBP1 knockdown arrested cells in the G0/G1 phase. G Heatmap showing differential gene expression in BXPC3 and Patu8988 cells under control conditions, olaparib treatment, TOPBP1 knockdown, or both. H Heatmap of gene set enrichment analysis (GSEA) used to analyze differences in KEGG signaling pathway enrichment among BXPC3 and Patu8988 cells treated with olaparib, TOPBP1 knockdown, or both. The P53 signaling pathway was upregulated in the TOPBP1-knockdown Patu8988 cells treated with olaparib, but not in BXPC3 cells

Fig 5: TOPBP1 expression is upregulated in patients with PDAC and associated with higher histological grade and shorter survival durations. A Patients with high TOPBP1 expression levels have worse overall survival (OS) than those with low TOPBP1 expression levels (n = 172, p = 0.0083) based on datasets from The Cancer Genome Atlas (TCGA). P-values were derived from the Kaplan–Meier survival analysis. B Patients with high TOPBP1 expression levels have worse OS than those with low TOPBP1 expression levels based on the Gene Expression Omnibus (GEO) datasets (n = 65, p = 0.038). P-values were derived from the Kaplan–Meier survival analysis. C TOPBP1 expression is higher in PDAC tissues with G3–G4 pathological stage compared to G1–G2 pathological stage in TCGA datasets (n = 179, paired t-test, p = 0.003). D Immunohistochemistry (IHC) showing TOPBP1 expression predominantly localized to pancreatic ductal tumor cells within the tumor. E Quantitative real-time PCR analysis of TOPBP1 expression in 58 pairs of PDAC tissues (tumor) compared to normal tissues (normal). F TOPBP1 expression levels in PDAC tissues based on IHC scores. G Patients with high TOPBP1 expression levels had a worse prognosis (OS) than those with low TOPBP1 levels (n = 47, Kaplan–Meier survival analysis, p < 0.0001) based on our center's patient data. H TOPBP1 expression in various PDAC cell lines analyzed by western blotting. I TOPBP1 expression in various PDAC cell lines analyzed by western blotting. Data shown are the mean ± SEM. Statistical analyses were performed with one-way ANOVA (*p < 0.05 and **p < 0.01 versus control)