Fig. 3.

Effects of uPAR silencing on Rho-GTPases activation, invasion and capillary morphogenesis in mesenchymal, amoeboid conditions. a The upper panel shows quantitative Real-Time PCR of uPAR relative expression in both cell lines after siPLAUR treatment. Not-targeting siRNA pool constructs were used as negative control (siCONTROL). The lower panel shows western blotting analysis of uPAR for each cell line after siPLAUR treatment. Dharmafect: treatment of cells with the transfection reagent alone. Numbers on the left of each Western blotting refer to molecular weights expressed in kDa. b Western blotting of total and GTP-loaded forms of small Rho-GTPasesRhoA and Rac1 under mesenchymal and amoeboid conditions in ECFCs untreated and treated with siCTRL/siPLAUR, respectively. Histograms report RhoA-GTP/RhoA and Rac1-GTP/Rac1 ratio obtained by band densitometry quantification. The same experiment on HMVECs gave similar results (not shown). c Matrigel invasion under mesenchymal and amoeboid conditions untreated and treated with siCTRL/siPLAUR, respectively. Histograms refer to quantification of Matrigel invasion assay obtained by counting the total number of migrated cells/filter. d In vitro angiogenesis before and after uPAR silencing by siPLAUR was measured by capillary morphogenesis at 6 h in the presence and in the absence of the protease inhibitor MIX. 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. 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