Fyn is essential for the PECAM-1 mechanoresponse in cell models. (A) Controls for siRNA experiments. ECs cultured on an elastic substrate were transiently transfected with Lipofectamine 2000 alone, negative control siRNA, or scrambled siRNAs, stretched in the presence of Na3VO4 for 10 min, and PECAM-1 tyrosine phosphorylation was assayed (top). Compared with unstretched ECs under each condition, stretched cells exhibited increased PECAM-1 phosphorylation by 1.42 ± 0.08- (LF only; n = 6), 1.84 ± 0.15- (control siRNA; n = 4), 1.69 ± 0.17- (scrambled siRNA-2; n = 3), and 1.49 ± 0.09- (scrambled siRNA-1; n = 3) fold (mean ± SEM). Student's t test was used to compare stretched to unstretched values. *, P = 0.0002 (LF control), 0.001 (control siRNA), 0.047 (scrambled siRNA-2), and 0.004 (scrambled siRNA-1). The bottom shows the expression of PECAM-1, Src, Yes, and Fyn in whole cell lysates by immunoblotting using actin as a loading control. Although not depicted here, Fer expression was not altered by these control siRNAs. (B–F) ECs cultured on an elastic substrate were transiently transfected with Src, Yes, or Fyn siRNA or Lipofectamine 2000 alone, and cell models were made after they formed confluent monolayers. Models were stretched or left unstretched for 5 min at 37°C in the presence of ATP and Na3VO4, and PECAM-1 phosphorylation was assayed. Each kinase associated with the cell model was checked by immunoblotting, using the level of actin as a loading control. (B and C) PECAM-1 phosphorylation was not suppressed in HAEC models with reduced Src expression. PECAM-1 phosphorylation levels are shown relative to unstretched models (mean ± SEM; n = 3). Student's t test was used to compare stretched to unstretched values. *, P = 0.0124 (LF control) and 0.0292 (Src siRNA). (D) PECAM-1 phosphorylation was not suppressed in BAEC models with reduced Yes expression. (E) Suppressed PECAM-1 phosphorylation in BAEC models with reduced Fyn expression is shown. (F) Levels of PECAM-1 phosphorylation in Yes and Fyn siRNA experiments are quantified and shown relative to unstretched models (mean ± SEM; n = 5). Student's t test was used to compare stretched to unstretched values. *, P = 1.7 ×10−7 (LF control) and 0.0032 (Yes siRNA); #, P = 0.1665. Black lines indicate that intervening lanes have been spliced out.
