Figure 7

HUVEC are sensitive to DMOG. (A) HUVEC were organized into spheroids and plated on collagen IV-coated plates in the presence or absence of 1 mM DMOG. F-actin and VE-cadherin were visualized by PromoFluor phalloidin staining and indirect immunofluorescence, respectively. Scale bar: 20 μm. (B) F-actin structures within the spheroids were visualized by apotome technique. Scale bar: 20 μm. Images were merged to show the height of the spheroids in z-axis. (D) Higher magnifications of F-actin fibers and VE-cadherin structures as shown in (Figure 7A). Scale bar: 20 μm. (C) After 24 h, nuclei were stained with Hoechst and the number of migrated cells was counted using ImageJ. Cell numbers were quantified in 35 spheroids of 5 different preparations. The graph summarizes the data of 20 spheroids of three different isolates (means ± SD). The area covered by F-actin stained cells (means ± SD) was determined in 35 spheroids of 5 different isolates. In each experiment the mean value of control spheroids was set to 1, error bars reflecting the variability within one experiment . *** p < 0.0001, Student’s t-test. (E) HUVEC were cultured in cell culture dishes treated with DMOG (1 mM) for 6 and 24 h. Phosphorylated MYPT was detected by Western blotting. Data are means ± SD of 3 experiments performed in duplicate. In each experiment the mean value of controls was set to 1, error bars of controls reflecting variability of biological samples. ** p < 0.01, Student’s t-test. (F) HUVEC cultured in cell culture dishes were treated with DMOG (1 mM) for 24 h. GTP-Rac was precipitated from 700 μg cellular protein. Total Rac was detected in 25 μg cellular protein. The blot is representative of 2 precipitations.