Fig. 4.

Par-4 and miR-200c upregulation reverse the HRas-mediated transformation in Panc-1 cells. (A) Cell scattering ability of HRas-Panc-1 cells subjected to Vehicle/NGD16 treatment or transfected with GFP/GFP-Par-4/Mock/miR-200c is depicted. (B) Western blot analysis of HRas-Panc-1 cells, subjected to Vehicle/NGD16 treatment or transfected with GFP/GFP-Par-4/Mock/miR-200c, for the ZEB-1 protein levels. (C) The bar-graph represents the relative ZEB-1 protein expression as quantified by the densiometric analysis. (D) Immunocytochemistry analysis for the ZEB-1 (red) expression in the HRas-Panc-1 cells transiently transfected with GFP/GFP-Par-4 (green), along with DAPI (blue). Photographs were taken at 20× magnification. Scale bar 10 μm. (E) Immunocytochemistry analysis for FAS trafficking (20× magnification) and ZEB-1 (20× magnification) expression in HRas-Panc-1 cells treated with Vehicle/NGD16. Scale bar, 50 μm and 20 μm respectively. (F) Western blot analysis of HRas-Panc-1 cells, subjected to Vehicle/NGD16 treatment for the Cleaved Caspase-3 protein levels. (G) Percent Cell viability of the GFP/GFP-Par-4-transfected Panc-1-GR10 cells stimulated with different concentrations of Gemcitabine (0, 1, 10, and 100 μM). (H) SEM images of Panc-1-GR10 cells transfected with GFP/GFP-Par-4, 1000× magnification. Scale bar 10 μm. (I) The bar graph represents the relative fold increase in the miR-200c level in the NGD16-treated tumor sample compared to the control. (J) Western blotting of the tissue lysates obtained from NGD16-treated and control-treated tumor samples for Par-4, E-cadherin, ZEB-1, and Vimentin expression analysis. Beta-actin was used as a loading control. (K) The schematic diagram depicts the mechanism of miR-200c induction by Par-4 resulting in EMT suppression. Data is represented as means ± S.D. of at least three independent experiments. *P < 0.05, **P < 0.01. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)