Figure 4

RhoA - Rho kinase signaling is active in DMOG-treated cells. (A) glEND.2 microvascular cells cultured in cell culture dishes were transfected with constitutively active eGFP-V14-RhoA. After 24 h, cells were stained for F-actin. Transfected cells are characterized by high density of F-actin fibers. Scale bar: 20 μm. (B) Spheroids were treated with DMOG (1 mM) or DMOG plus H1152 (0.75 μM) for 24 h. The Rho kinase inhibitor reduced the size of the remaining spheroids and prevented stress fiber formation. Nuclei were stained with Hoechst, and F-actin fibers with PromoFluor phalloidin. Scale bar upper panel: 100 μm; lower panel: 20 μm. (C) The number of cells migrated off spheroids was determined in two independent experiments with at least 15 spheroids for each condition. Mean cell numbers (means ± SD) of control cells was set to 1, error bars of control values represent variability within one experiment. ** p < 0.01 as indicated, *** p < 0.001 compared to respective controls, n.s: not significant; ANOVA with Dunnett’s multiple comparison test. (D) Cells cultured in cell culture dishes were incubated with DMOG for 6 and 24 h. Phosphorylated MYPT was detected in cell culture homogenates by Western blotting. The blot is representative of three experiments. (E) glEND2 cells stably transfected with GFP, shHIF-1α or shHIF-2α were cultured in cell culture dishes with (D) or without (C) DMOG for 24 h. Phosphorylated MYPT was detected by Western blotting. For quantification, the ratio pMYPT/Vinc of non-treated cells was set to 1 at each experimental condition (Co). ** p < 0.01, n = 3, means ± SD, ANOVA with Tukey’s multiple comparison test.