sVEGFR1-ex12 contributes to FGF-2 pro-angiogenic functions in endothelial cells. a The schedule sketch indicates the protocol used to generate sVEGFR1-i13 or sVEGFR1-ex12 knockeddown HUVEC-RFP cells by siRNA and to collect endothelial cells for xCELLigence (b) or 3D invasion (c) assay, respectively. bUpper left panel: sVEGFR1-i13 and sVEGFR1-ex12 mRNA levels were quantified by RT-qPCR in HUVEC-RFP transfected with either control siRNA (mis, black bars) or with a mixture (50:50) of two distinct siRNA against either sVEGFR1-i13 (white bars) or sVEGFR1-ex12 (grey bars). GAPDH was used as an internal control. Mean ± SD are presented (n=4; 2 technical replicates of 2 independent experiments, t test, *p<0.05, **p<0.01, ***p<0.001). Lower left panel: cellular viability quantified by using trypan blue exclusion counting in HUVEC-RFP just before plating for xCELLigence assay (mean ± SD, n=3; unpaired t test, ns: not significant). Right panel: xCELLigence assay was used to test FGF-2 (3nM) effects on adhesion/proliferation/survival of sVEGFR1-i13 or sVEGFR1-Ex12 depleted HUVEC-RFP (n=3). Mismatch siRNA was used as a positive control to ensure FGF-2 protective effects on cell viability in this experimental design. c 3D invasion assay in collagen I gels. Left panels: mosaic of confocal images covering around 300 μm of matrices border for HUVEC-RFP transfected with the indicated siRNA. HUVEC-RFP cells are in red and nuclei in blue. Scale bar = 100μm. Right panels: bar charts of the quantification of the invading distance of HUVEC-RFP within the 3D collagen matrices and the number of invading cells (nuclei) per 300 μm of gel border. Each black dot indicates one invading sprout. Graphs represent mean values ± SD (n=3; t test, *p<0.05, **p<0.01). dLeft panels: confocal illustrations of the impact of the peptide p12 that blocks the interaction between sVEGFR1 and β1 integrin or a control scramble peptide on FGF-2-mediated HUVEC-RFP sprouting/invasion in collagen I gels (n=3). Scale bar is 100 μm. Right panels: Bar charts of the quantification of invading distance and the number of invading cells as indicated above (mean values ± SD, unpaired t test, *p<0.05, ns: not significant)
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