Fig 2: Pin1 binds to p65 and facilitates NF‐κB activation in pancreatic cancer cells. (A) Co‐immunoprecipitation analysis of the interaction between Pin1 and p65 in pancreatic cancer cells. (B) Double immunofluorescent staining revealed co‐localization of the Pin1 and p65 proteins in Capan‐1 and SW19990 cells (scale bar, 5 μm). (C) The effects of Pin1 knockdown on phosphorylation of p65 were assessed by Western blot. (D) Nuclear translocation of p65 induced by Pin1. MIA PaCa‐2 cells were transfected with Pin1 or empty vector, and p65 location was determined (scale bar, 25 μm). (E) Increase of NF‐κB by Pin1 upregulation. Cells were cotransfected with Pin1 vector or control and NF‐κB‐Luc construct, followed by luciferase assay

Fig 3: Inhibition of Pin1 expression suppressed tumor cell proliferation in zebrafish. (A) Combined fluorescence and bright field images of zebrafish xenograft model transplanted with CNE1-LV3 or CNE1-shPin1 into the perivitelline space around the yolk sac at 1, 3, 5, and 7 dpt, respectively. The relative fluorescence intensity of the tumor cells in zebrafish was quantified. The amount of tumor cells at 1 dpt was used as the baseline with which the amount of tumor cells at other time points was compared. The differences in cell proliferation between the two groups were quantitatively analyzed by two-way ANOVA (CNE1-LV3 group, n = 6 transplanted fishes analyzed; CNE1-shPin1 group, n = 11 transplanted fishes analyzed). Scale bar: 1 mm. (B) Whole-mount embryo immunofluorescence staining of Pin1 and Cyclin D1 expression in zebrafish with tumor cells transplanted at 7 dpt. Selected areas of images (white dashed line) are shown with a higher magnification below. Scale bar: 50 μm; 10 μm in the amplified image. The mean fluorescence intensity of Pin1 and Cyclin D1 was quantitatively measured, and the data were quantitatively analyzed by Student’s t test (CNE1-LV3 group, n = 6 transplanted fishes analyzed; CNE1-shPin1 group, n = 4 transplanted fishes analyzed). Data are shown as mean ± SD. dpt: days post-transplantation; EGFP: enhanced green fluorescent protein. **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig 4: Pin1 promotes and participates in IL‐18‐induced oncogenic behaviour in pancreatic cancer cells. (A) Pin1‐silenced Capan‐1 and SW1990 cells both exhibited significantly decreased cell motility, while Pin1‐overexpressed MIA PaCa‐2 cells dramatically increased cell motility in the wound healing assay (scale bar, 100 μm); quantitation of the data is shown in (B). (C) The expression of IL‐18 was determined by Western blot. (D) A CCK‐8 assay was used to detect the proliferation of MIA PaCa2 cells overexpressing Pin1 and Pin1‐overexpressing cells transfected with IL‐18 siRNA. Knockdown of IL‐18 by siRNA reversed the proliferation ability enhanced by Pin1 overexpression. (E) Transwell assays were applied to measure the migration and invasiveness of MIA PaCa2 cells overexpressing Pin1 and transfected with IL‐18 siRNA. Knockdown of IL‐18 by siRNA decreased both the capacity of migration and invasion induced by Pin1 overexpression; quantitation of the data is shown in (F). (G) Pin1 knockdown cells and scrambled control were treated with rhIL‐18 (20 ng/mL) or PBS for 36 h and subjected to the CCK‐8 assay. (H) Pancreatic cancer cells were treated with rhIL‐18 (20 ng/mL) or PBS for 36 h and subjected to the transwell assay. Knockdown of Pin1 reversed the capacity of migration and invasion induced by IL‐18, while IL‐18 saved the capacity of migration and invasion decreased by Pin1 silencing (scale bar, 200 μm). (I) The relative cell number of migration was quantitated and shown as means ± SD. *P < .05, **P < .01

Fig 5: Schematic illustration summarizing the Pin1‐NFκB‐IL‐18 feedback loop in pancreatic cancer cells