Effect of ectopic SMAD3 expression on cell migration following RNAi-mediated depletion of endogenous TGFβ1. Panc1 or MDA-MB-231 cells were transfected with 50 nM each of either ctrl siRNA or TGFB1 siRNA in combination with either empty pcDNA3.1 vector or pcDNA3.1 containing a SMAD3 cDNA. Forty-eight h after transfection cells were analyzed by real-time cell migration assay. Data are the means ± SD of parallel 3-4 wells and are representative of 3 independent experiments. The differences between curves D and B were first significant (p < 0.05) at 3:30 (Panc1) or 3:00 (MDA-MB-231), and for Panc1 cells remained significant over the 24-h assay period. A transient effect of ectopic SMAD3 was also seen in control conditions (curves A vs. curves C, significant between time points 6:30 and 13:00 (Panc1) and 4:00 and 7:30 (MDA-MB-231) in accordance with previous findings [18]). KD of TGFB1 or overexpression of SMAD3 was verified by immunoblotting (insets), siCo, ctrl siRNA; siTB1, TGFB1 siRNA; V, empty vector; pS3, SMAD3 expression vector.