Fig. 7
Rab5c modulates CD93-mediated EC motility. HUVECs were transduced with lentiviral particles expressing unrelated (unr) or Rab5c shRNAs (clone 31 and 33). a: Cell lysates from shRNA expressing ECs were analyzed by Western blotting using antibodies against Rab5a, Rab5b, or Rab5c. Anti-β-actin antibodies were used to confirm equal loading. b: Representative images of control (shRNA unr) and Rab5c-silenced (shRNA Rab5c, clone 33) HUVECs at early and late phases of spreading. Cells were analyzed by confocal microscopy using anti-CD93 and anti-Rab5c antibodies. Overlay of stained cells is shown. Dashed lines indicate cell boundaries. Scale bars, 6 μm. c: Quantitative analysis of CD93+ve vesicles per cell from late spreading ECs treated as in b. The vesicle numbers represent the mean ± SD of three independent experiments (n = 20 cells). ****P < 0.0001; unpaired t-test. d: Flow cytometry analysis of CD93 plasma membrane levels in control (unr) and Rab5c-silenced (clones 31 and 33) HUVECs. Cells stained only with the secondary antibody are shown (ctr). The mean fluorescence intensity (MFI) is reported. Total cell extracts from the same shRNA expressing cells were analyzed by Western blotting using anti-CD93 and anti-Rab5c antibodies. Anti-β-actin antibodies were used to confirm equal loading. e, f: Representative images of the confocal microscopy analyses of control (shRNA unr) and Rab5c-silenced (shRNA Rab5c, clone 33) HUVECs at 5 h after production of a double-sided scratch in the cell monolayer. Phalloidin and anti-CD93 (e) or anti-β1 integrin (12G10) (f) antibodies were used to image cells. Arrows indicate direction of migration and dotted lines indicate the migrating front. Overlay of stained cells is shown. Scale bars, 40 μm. Magnifications of the squared areas are shown as wdc images. In wdc images, scale bars are 15 μm. In b, e, and f, same results were obtained when using the clone 31 for Rab5c knockdown. g: Quantification of CD93 and active β1 integrin along the migrating front areas indicated by dotted lines in e and f. Bars represent the percentage of fluorescence intensity of the control. Data are presented as the mean ± SD of three independent experiments (n = 5 different areas along the migrating edge). **P < 0.01; unpaired t-test. h: Representative images of wound closure in HUVECs transduced with lentiviral particles expressing control (unr) or Rab5c shRNAs. Cells were photographed at 0 and 8 h. Scale bar, 100 μm. i: The percentage of scratch area was calculated from images acquired at time 0 and 8 h following the wound. Analyses were performed using ImageJ. The graph represents the means ± SD, n = 6 images per condition pooled from two independent experiments. *P < 0.05; unpaired t-test