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Fig. 2 Reduction of the RhoA activity contributes to the R1881-reduced endothelial cell migration. (a) Treatment with R1881 (5 nM) for 6 h reduced the RhoA activity in HUVECs, and this effect was abolished by pre-treatment with HF (5 nM). Top panel: a representative result of the level of RhoA-GTP in HUVECs. Bottom panel: quantitative results of RhoA-GTP after adjust with their own input. Values present the means of fold of corresponding control ± s.e.mean. (n = 4). *p ＜ 0.05 different from corresponding control. #p ＜ 0.05 different from the R1881-treated group. (b). Transfection with RhoA V14 pcDNA prevented the R1881-induced migration inhibition in HUVECs, and this prevention effect was abolished by pre-treatment with Y27632, a ROCK inhibitor. Values present the means of fold of corresponding control ± s.e.mean. (n = 3). *p ＜ 0.05 different from corresponding control. #p ＜ 0.05 different from the R1881-treated group. (c) Treatment with R1881 (5 nM) for 6 h increased the level of cytosolic p27 protein, but reduced the level of cytosolic RhoA protein in HUVECs. Values shown in parentheses represent the quantified results after adjusted with their own GAPDH protein levels and expressed as fold of control. (n = 3). (d) Treatment with R1881 (5 nM) for 6 h increased the formation of RhoA-p27 complex in HUVECs. (e) Co-immunoprecipitation analysis demonstrated that treatment with R1881 (5 nM) for 6 h increased the RhoA ubiquitination in HUVECs. Values present the means of fold of corresponding control ± s.e.mean. (n = 3). *p ＜ 0.05 different from the DMSO-treated group. (f) Treatment with R1881 (5 nM) for 6 h reduced the level of RhoA protein, and this reduction was abolished by pre-treatment with MG132, a proteasome inhibitor. Values shown in parentheses represent the quantitative results after adjusted with their own GAPDH protein levels and expressed as fold of control. (n = 3). (g) Transfection with p27 siRNA, which knocked-down p27 expression (top panel), abolished the R1881-induced migration inhibition in HUVECs. Values present the means of fold of corresponding control ± s.e.mean. (n = 4). *p ＜ 0.05 different from the DMSO-treated group. #p ＜ 0.05 different from the R1881-treated group. HF, hydroxyflutamide; R1881, metribolone.