Fig. 5

VEGF role in amoeboid angiogenesis. a Confocal microscopy for F-actin by phalloidin staining (red fluorescence) and VEGFRII (green fluorescence) under mesenchymal (-MIX) and amoeboid (+MIX) conditions, in the absence and in the presence of VEGF. Magnification: 40 X. Phalloidin was used to make more evident the cell membrane profile under amoeboid conditions. b Histogram on the left refers to quantification of Matrigel invasion assay obtained by counting the total number of migrated cells/filter. The assay was performed in the presence and in the absence of the MIX added to the Matrigel solution before polymerization and after addition of VEGF in the cell suspension. On the right capillary morphogenesis performed at the same conditions described for Matrigel invasion assay. Numbers on the lower right side of each picture indicate the percent field occupancy of capillary plexus as described in the Materials and Methods section. Quantification was performed at 6 h after seeding and was obtained by scanning of six to nine photographic fields for each condition. c Hystogram on the left shows results from boyden chamber invasion assay through a thick Matrigel coating in mesenchymal and amoeboid conditions, before and after uPAR silencing and with and without VEGF stimulation. siCTRL: negative control. siPLAUR: specific siRNA smart pools directed to uPAR gene. On the right capillary morphogenesis performed at the same conditions described for Matrigel invasion assay. d Western blotting results show the effects of VEGF, in mesenchymal and amoeboid conditions, on the intracellular signaling molecules RhoA and Rac1, the phosphorylation of KDR and MLC2 and WAVE2. Numbers on the left refer to molecular weights expressed in kDa. Histograms report band densitometry. Results are the mean of 5 different experiments performed in duplicate, on two different clones derived from two different donors, on each cell line and are shown as mean value ± SD. *: p < 0.05; **: p < 0,001; ***p < 0,0001 significantly different from control. Figure 5 shows results obtained with ECFCs. HMVECs gave similar results (not shown)