Fig. 1.

RhoC promotes proliferation and negatively regulates migration through activation of VEGF. (A) Serum-starved HUVECs were stimulated with 10 ng/ml VEGF-A for 1, 3 and 5 min. Lysates were immunoprecipitated with the respective substrate GST-tagged beads, and GTP-bound RhoC and GTP-bound RhoA were detected by immunoblotting. (B) HUVECs were serum starved overnight and stimulated without (−V) or with VEGF-A for 2 or 5 min (+V2 and +V5, respectively). Lysates were immunoprecipitated with GST-tagged beads for the respective substrate, and GTP-bound RhoC and RhoA were detected by immunoblotting. A densitometry analysis of the depicted immunoblots was performed using ImageJ software and is shown in the graphs below the blots. (C) HUVECs were transfected with control or RhoC siRNA using Oligofectamine for 48 h. 4×10⁴ cells were plated in a 24-well plate, serum starved (0.2%) overnight and treated with 10 ng/ml VEGF-A. Thymidine incorporation assays were performed. ***P≤0.0001, paired two-tailed Student's t-test (RhoC siRNA +VEGF treated group versus control siRNA +VEGF treated group); *P≤0.05 (RhoC −VEGF group versus control siRNA −VEGF group). (D) 5×10⁴ serum-starved HUVECs treated with control or RhoC siRNA were seeded into collagen-coated Transwell chambers and inserted into 24-well plates containing low-serum EGM. 10 ng/ml VEGF-A was added in the lower chamber and a Transwell migration assay was performed for 4 h. ***P≤0.0001, paired two-tailed Student's t-test (RhoC siRNA +VEGF treated group versus control siRNA +VEGF treated group). Experiments were repeated at least three times, and graphs in C and D show the mean±s.d.