Fig 2: Relationship between PAUF and TLR4 expression in ovarian cancer cell lines. (A) To probe surface and intracellular TLR4 expression level in SKOV3 and A2780 cells, PE-conjugated TLR4 antibody was confirmed and used in flow cytometric analysis. (B,C) PAUF expression level and secretion in ovarian cancer cells (SKOV3, A2780) were detected using western blot analysis, and PAUF in the culture supernatant of cancer cells was detected using ELISA. (D) The expression level of intracellular TLR4 was assessed by flow cytometric assays. TLR4 expression was decreased significantly in both cells after transfection of two TLR4-siRNAs in comparison to that of control-siRNA or control. (MFI: Mean Fluorescence Intensity) (E) To determine activation of MAPKs (ERK, P38 and JNK) and AKT by PAUF, starved cancer cells were treated with or without recombinant PAUF (5 μg) and analyzed by western blotting. (F) To confirm MAPK activation in siRNA transfected A2780 or SKOV3 cells, cells were transfected with TLR4-siRNA or control siRNA and starved for 16 hours. Cells were treated with PAUF (5 μg) for 20 min and analyzed by western blotting. The number below each western blot represents the ratio of the intensity of the band over the control intensity of scramble siRNA-treated cells. (G) For the cell proliferation assay, control, TLR4-, or PAUF-siRNA transfected A2780 or SKOV3 cells were cultured in 96-well white plates, and cell proliferation was detected using a Cell Titer-Glo luminescence assay kit. The data shown are the means ± s.e.m. for three independent experiments. β-actin was used as an internal reference. *p < 0.05; **p < 0.01. The gels images were cropped and full-length gels and blots are included in the Supplementary Figure S6.

Fig 3: The associations between PAUF and TLR4 in patients with epithelial ovarian cancer. (A) TLR4 expression was positively correlated with expression of PAUF (r = 0.256, p = 0.001). (B) PAUF expression showed significant positive correlations with TLR4 expression in advanced stage (stage III/IV; r = 0.279, p = 0.002) and grade 3 (r = 0.346, p = 0.001).

Fig 4: Expression of PAUF and TLR4 in patients with epithelial ovarian cancer. Expression of PAUF and TLR4 was analyzed in specimens from normal tissue (n = 72), benign tumor (n = 69), borderline tumor (n = 62), ovarian cancer (n = 205), and metastatic tissues (n = 52). The histoscores were computed based on intensity and tissue area of positive staining. PAUF (A) and TLR4 (B) expression increased during tumor progression. PAUF and TLR4 expression was compared between specimens from chemosensitive (n = 147) and chemoresistant (n = 43) ovarian cancer. Cancer patients who experienced recurrence within 6 months after platinum and paclitaxel chemotherapy were considered resistant. PAUF (C) and TLR4 (D) expression levels were higher in chemoresistant tumors (p = 0.017 and p = 0.001, respectively).

Fig 5: Representative images of immunohistochemical staining of pancreatic adenocarcinoma upregulated factor (PAUF) and toll-like receptor 4 (TLR4) in human ovarian cancer specimens. The top row (A,B) represents normal ovarian tissue, middle row (C,D) shows immunoreactivity of ovarian carcinomas and the bottom row (E,F) indicates immunonegativity of ovarian caricinoma. The stromal cells of normal ovarian tissue show immunonegativity for PAUF and TLR4. The scale bar represents 100 μm.