Figure 3

A multi-faceted siRNA screen to test predicted node knockdown combinations in vitro. (a) Schematic of the experimental design of the screen to test node knockdown combinations that are predicted to inhibit TGFβ-driven EMT. At 0 h, epithelial-like Huh7 cells were transfected with siRNA combinations or a scrambled siRNA control (2 nM per siRNA), and then plated for harvesting of protein, mRNA, and cell migration. At 45 h post transfection, cells were serum starved for 3 h. At 48 h, cells were treated with TGFβ (5 ng/ml) or a vehicle control for an additional 48 h. At 96 h, cells were harvested for mRNA and protein expression. An aliquot of siRNA-transfected cells were plated at 0 h in 96-well plates for assessment of migration with the Oris Cell Migration Assay (see Materials and Methods for details). These cells were treated with either TGFβ or a vehicle control at 48 h. Simultaneously, migration stoppers were removed at 48 h post transfection and analysis of cell migration was performed at 48, 72, and 96 h post transfection. (b) The effect of model-predicted node knockout combinations on E-cadherin expression in TGFβ-treated cells relative to vehicle control treated cells, as measured by quantitative immunoblotting. (c) The effect of model-predicted node knockout combinations on in vitro cell migration in TGFβ-treated cells relative to vehicle control treated cells. The percent change in TGFβ-driven migration (top) and heat-map of TGFβ-driven migration (bottom) relative to a scrambled siRNA control 24 and 48 h after TGFβ treatment. EMT, Epithelial-to-mesenchymal transition, mRNA, messenger RNA; siRNA, small interfering RNA, TGFβ, transforming growth factor beta.