Figure 3.

Hypoxia‐driven paracrine OPN signaling promotes EMT and CSC‐like properties in PCCs. (A) The mRNA expression levels of OPN, the αv subunit of integrin, and the β3 subunit of integrin in 4 pancreatic cancer cell lines and PSCs were analyzed by quantitative real‐time RT‐PCR. (B and C) The protein expression levels of OPN, the αv subunit of integrin, and the β3 subunit of integrin in 4 pancreatic cancer cell lines and PSCs were determined by western blot analysis. β‐Actin was used as an internal control. (D) The cell supernatants of 4 pancreatic cancer cell lines and PSCs were collected, and the OPN concentration was measured by ELISA. (E) Representative images from the wound healing assay after rhOPN, PSC‐CM, or PSC‐CM + OPN Ab treatments in BxPC‐3 and CFPAC‐1 cells. Images were obtained at 0 h and 24 h. The scale bar represents 100 μm. (F and G) Representative images of the Matrigel invasion assay after rhOPN, PSC‐CM, or PSC‐CM + OPN Ab treatments in BxPC‐3 and CFPAC‐1 cells. The invasive cells were quantified by counting the number of cells in 10 random fields. The scale bar represents 50 μm. (H, I and J) Representative images of the tumorsphere formation assay after rhOPN, PSC‐CM, or PSC‐CM + OPN Ab treatments in BxPC‐3 and CFPAC‐1 cells. The number of tumorspheres was counted and plotted, and the percentage of tumorspheres with diameters of 50–100 μm, 100–150 μm, or >150 μm was calculated and plotted. The scale bar represents 50 μm. (K) The protein expression levels of EMT and CSC markers after rhOPN, PSC‐CM, or PSC‐CM + OPN Ab treatments were determined by western blot analysis in BxPC‐3 and CFPAC‐1 cells. β‐Actin was used as an internal control. Q‐PSC: Quiescent pancreatic stellate cell. A‐PSC: Activated pancreatic stellate cell. All data are shown as the mean ± SD of at least three independent experiments. One‐way ANOVA and Tukey's multiple comparisons test were performed to determine significance. *P < 0.05, **P < 0.01, ns: no significance.