Figure 5

The role of FLASH in EMT progression. (a) PANC-1 cells were transfected with a pool of siRNA duplexes targeting FLASH and treated with 100 ng/ml TGFβ for the indicated periods of time. E-cadherin (top panel) and ZEB1 (middle panel) protein levels in siRNA- and mock-transfected cells treated with TGF-β or left untreated were determined by western blot analysis and the expression normalized to actin loading control (bottom panel). (b) Relative mRNA levels of E-cadherin (fold increase, left graph) and FLASH (right graph) were determined by quantitative PCR (qPCR). The graphs represent the average of three independent experiments. The significance of differences was determined by Student's t-test (**P<0.0025). (c) PANC-1 cells were transfected with a pool of siRNA duplexes targeting FLASH and treated with TGFβ (100 ng/ml) for 1 h. Phosphorylated SMAD2 and total SMAD2 protein levels in mock-transfected and FLASH-depleted cells treated with TGFβ was determined by western blot. (d) ZEB1, Snail and Slug mRNA expression in mock-transfected and FLASH-depleted cells as detected by qPCR. The graphs represent the average of three independent experiments. The significance of differences was determined by Student's t-test (**P<0.0025). NS, not significant.