Fig 2: Signaling pathways in Pkd2 deficiency osteoblasts.(A) Basal intracellular calcium ([Ca2+]i) levels. Heterozygous Pkd2 +/null osteoblasts (n = 6) showed a significantly lower basal [Ca2+]i levels compared with wild-type Pkd2 +/+ osteoblasts (n = 6), and homozygous Pkd2 null/null osteoblasts (n = 6) had greater reductions of basal [Ca2+]i compared with the heterozygous Pkd2 +/null osteoblasts (n = 6). (B) Flow-induced [Ca2+]i response. A gene dose-dependent reduction of flow-induced [Ca2+]i response was observed in the Pkd2-deficient osteoblasts compared with wild-type Pkd2 +/+ osteoblasts, indicating an impairment of calcium channel activity in the Pkd2-deficient osteoblasts. (C) TAZ/YAP-dependent transcriptional activation as assessed by 8xGTIIc-luciferase activity. A gene dose-dependent reduction of basal TAZ/YAP activity was observed the in immortalized primary osteoblasts derived from the Pkd2-deficient osteoblasts compared to the wild-type controls. (D) Western blot analysis. Phosphorylation of YAP was significantly increased, while the amount of TAZ was significantly decreased in Pkd2-deficient osteoblasts than in wild-type controls. Data are mean±S.D. from triple independent experiments. Values sharing the same superscript in different group are not significantly different at P<0.05.

Fig 3: Knockdown of gga-miR-375 promotes proliferation and the cell cycle in DF-1 cells. (A) DF-1 cells transfected with anti-gga-miR-375 had decreased gga-miR-375 mRNA expression at 48 h following transcription. Anti-gga-miR-con group and mock group were used as the controls. DF-1 cells transfected with anti-gga-miR-375 exhibited increased (B) YAP1 protein and (C) mRNA expression levels compared with the control groups. (D) Overexpression of anti-gga-miR-375 promotes cell proliferation as indicated by Cell Counting Kit-8 assay results 24, 48 and 72 h after transfection. (E) Overexpression of anti-gga-miR-375 promotes the cell cycle as demonstrated by flow cytometry results. (F) DF-1 cells transfected with anti-gga-miR-375 had upregulated expression levels of YAP1, cyclin D1 and cyclin E. Anti-gga-miR-con and mock groups were used as the control groups. Data are presented as the mean ± SD of three independent experiments. **P<0.01 vs. anti-miR-con group. miR, microRNA; YAP1, Yes-associated protein 1; NC, negative control; con, control; OD, optical density.

Fig 4: Nuclear expression of YAP1 in GBC is the second most important prognostic factor after depth of invasion in patients with subserosal infiltration (pT2). (A) GBC patient survival rates by tumor infiltration. Subserosal infiltration had an intermediate prognostic value (median survival 19.9 months) by univariate analysis; (B) Subserosal gallbladder cancer patient survival rates by high and low YAP1 nuclear expression. Patients with subserosal GBC and high nuclear YAP1 had a median survival of 8.0 months compared to patients with subserosal GBC and low nuclear YAP1, which have undefined survival by multiple correlation analyses. MS: Median survival; HR: Hazard ratio. 95% Confidence interval (0.80–1.86 months).

Fig 5: In vitro treatment with Verteporfin reduces migration and invasion capabilities in GB-d1 and G-415 cell lines. (A) Reduced expression of YAP1 and phospho-YAP in GB-d1 and G-415 cells treated with increasing concentrations of Verteporfin (VP). (B) Relative migration of GB-d1 and G-415. Magnification: 20×. (C) Relative invasion of GB-d1 and G-415. Results are represented as Mean ± SD from 3 independent experiments (* p < 0.05, ** p < 0.01 and ns: no significant by one-sample t-test). Magnification: 20×.