Fig. 5

Cell rounding and retraction fiber formation in response to FVIIa and ephrin-A1 is dependent on a RhoA/ROCK pathway. a MDA-MB-231 cells were pre-incubated with either DMSO or 25 or 50 μM PI3K inhibitor LY294002 before 10 nM FVIIa was added for 1 h. Samples were analyzed by Western blot. S897-phosphorylation was increased to 163.1 ± 30.2 % of control in DMSO-treated cells (p = 0.006), whereas 25 μM or 50 μM LY294002 abolished induction by FVIIa to 96.4 ± 8.6 % vs 77.1 ± 11.7 % (p = 0.08) or 89.9 ± 6.7 % vs 91.7 ± 8.1 % (p = 0.78), respectively. Values are expressed as % of control. N = 3–4, results are presented as means ± SD. b MDA-MB-231 cells were pretreated with 10 μM ROCK-inhibitor Y-27632 or 25 μM PI3K inhibitor LY294002 for 30 min and then stimulated as in Fig. 4. Y-27632 pre-treatment abolished the effects of ephrin-A1 as well as the potentiation by FVIIa (4.8 ± 1.3 % vs 4.6 ± 4.1 %, p = 0.93), while LY294002 had no effect (32.5 ± 7.3 % vs 14.6 ± 4.9 %, p = 0.007). N = 3–4, results are expressed as means ± SD of the percentage of rounded cells with retraction fibers. c RhoA expression was silenced by siRNA in MDA-MB-231 cells, and knock-down was verified by Western blot. In Scr-transfected cells FVIIa potentiated ephrin-A1 induced cell rounding (35.87 ± 14.80 vs 10.49 ± 4.29 %, p = 0.046), whereas RhoA knock-down cells were unresponsive to ephrin-A1 and FVIIa (3.30 ± 2.50 % vs 0.92 ± 0.88 %, p = 0.19). N = 3, results are expressed as means ± SD of the percentage of rounded cells with retraction fibers