Figure 3
Adipocytes sustain pancreatic preneoplastic lesions aggressiveness through a WNT paracrine network. (a) Western blot analysis for Vimentin, β-catenin, phpspoGSK3 and total GSK3 of HPDE and HPDE/KRAS cultured with hBMSCCM or adipocytes (ADIPOCM) treated or untreated with vantictumab. γ-Tubulin was used as loading control. The relative protein quantification, using ImageJ software, was performed and relative GSK3 and γ-tubulin ratio are indicated. (b) Cell proliferation assay of HPDE and HPDE/KRAS cell lines cultured with the conditional medium from hMBSC and adipocytes treated or untreated with vantictumab. The mean values and 95% confidence interval (CI) from three independent experiments done in quadruplicate are shown. (c) Bar histograms of cell migration levels of HPDE and HPDE/KRAS cell lines cultured with the conditioned medium from hMBSC and adipocytes treated or untreated with vantictumab. The mean values and 95% CI from three independent experiments done in quadruplicate are shown. ***P<0.001 by two-tailed unpaired Student’s t-tests. Photographs of the wound area were taken by using phase-contrast microscopy immediately at indicated time point after the incision. All these experiments were performed using hBMSC isolated from the healthy donor 1 (#1) and its relative adipocytes. (d) Transwelll invasion assay of HPDE and HPDE/KRAS cell lines cultured with the conditioned medium from adipocytes and hBMSC control cells with or without vantictumab. The invasion chambers were used for treated and untreated group. The values obtained were calculated by averaging the total number of the cells from three filters. All experiments were performed in triplicate. The mean values and 95% CI from three independent experiments done in quadruplicate are shown. ***P< 0.001, **P<0.005, *P<0.05 and NS (Not Significative), by one-way analysis of variance and post Dunnett's mutiple comparison post test using hBMSC as control.
