Fig. 6.
Rab4 regulates the angiogenic process independent of mTOR and p38 activation. Equivalent numbers of LMVEC were transiently transfected with GFP or Rab4Q67L cDNA. A: following 48 h, cell migration was measured by wound-healing assay in the presence or absence of SB203580 (10 nM, 30 min) preexposure followed by treatment with rapamycin (10 nM) and VEGF (50 ng/ml) up to 6 h. Images were captured and data was analyzed by using MiToBo analyzer in ImageJ and expressed as migration (μm) per hour. B: following 48 h, cells were replated onto gelatin-coated dishes and treated in the presence or absence of SB203580 (10 nM, 30 min) followed by treatment with rapamycin (10 nM) and VEGF (50 ng/ml) for 2 h. Cells adhered to gelatin were measured by MTT assay (O.D.570nm). C: transfected cells were plated directly onto Matrigel-coated wells for 48 h. Cells were then preexposed to SB203580 (10 nM, 30 min) followed by treatment with rapamycin (10 nM) and VEGF (50 ng/ml) for a further 6 h, images of tube formation were captured, and in vitro vessel formation was assessed by using Angiogenesis Analyzer in ImageJ and expressed as number of junctions formed. D: at 24 h prior to cDNA transfection, LMVEC were transiently transfected with p18 or nonsilencing siRNA. After a further 48 h, cells were replated onto gelatin-coated dishes and treated in the presence or absence of VEGF (50 ng/ml) for 2 h. Cells adhered to gelatin were measured by MTT assay (O.D.570nm). Data are presented as means ± SD; n = 4 (A), n = 4–5 (B), n = 6 (C); *P < 0.05 vs. vehicle, #P < 0.05 vs. GFP, †P < 0.05 vs. nonsilencing siRNA.