Figure 4
Effect of Rac1b depletion on EMT-associated genes. (A) Phase-contrast images of Panc1 cells transfected with an irrelevant control siRNA (siCo) or siRNA to Rac1b (siRac1b) and subsequently treated for 48 h or not (-) with 5 ng/ml TGF-β1 in medium with 0.5% FBS. Three experiments were performed in total, of which one is shown. The arrows point to cells with an elongated morphology. Insets indicate the percentage of spindle-shaped cells per visual field (mean ± SD, n = 3, independently counted by two investigators), p = 0.0039 for siCo + T vs. siRac1b + T and p = 0.0025 for siRac1b-T vs. siRac1b + T. Magnification: ×400. (B) Panc1 cells were transfected with an irrelevant control siRNA (Co), siRNA specific to Rac1b, or siRNA targetting both Rac1 and Rac1b. Transfected cells were treated with TGF-β1 for 24 or 48 h in medium containing 0.5% FBS and then subjected to sequential immunoblotting for E-Cadherin and Snail, Rac1 and Rac1b to verify functionality of the siRNAs, and HSP90 as loading control without intermittent stripping. The bands shown are all from the same blot/gel. Three experiments were performed in total of which one is shown. c Panc1 cells were transfected with Rac1b (R1b) or control (Co) siRNA, serum-starved and treated with TGF-β1 for 24 and 48 h. Cells were then subjected to qPCR-based expression analysis for the indicated genes. Slug was used as positive control9. Successful Rac1b depletion (21.4 ± 0.0069% of Co) was verified by qPCR using exon 3b-specific primers. Data are given as Relative (Rel.) mRNA expression and represent the mean ± SD from at least three independent experiments. Asterisks indicate significant differences.
