Sirna oligos

To examine the influence of Yap target genes on cell motility, we performed siRNA experiments in P19 embryonal carcinoma cells. siRNA oligos were obtained from Integrated DNA Technologies (Coralville, Iowa). The methods used for siRNA knockdown experiments have been described previously (8 (link)). After 24 hours of siRNA treatment, trypsinized cells were suspended in collagen gel mix (Millipore). Nylon mesh (20 μm) was applied to solidified collagen gel spots in 4-well plates, and cell/collagen mixes were dispensed to meshes. Plates were incubated at 37°C (5% CO₂) for 1 h and α-MEM culture media with 10% fetal bovine serum (FBS) was subsequently added. After 24 h of culture, the top gel and nylon mesh were removed, and the bottom gel was immmunostained accordingly. We examined the statistical significance of the differences between groups by using Mann-Whitney U tests. P<0.05 was considered significant.

For endogenous STAT3 knockdown assays, tumor cells (e.g. MCF-7 cells and E0771 cells) were transfected with designated 50nM siRNA oligos (purchased from Integrated DNA technologies and Sigma-Aldrich) in combination of Oligofectamine and Opti-MEM I reduced serum medium (Cat. #51985034) for 12 hours and then replaced with the RP10 medium. For chemical inhibition of STAT3, S3I-201 or NSC74859, specific STAT3 inhibitors, were added in the culture medium at doses from 0 to 25µM for 12 hours to inhibit STAT3 activation in tumor cells.