Fig. 2

FGF-2-mediated HUVEC survival requires SR proteins accumulation and phosphorylation. a HUVEC were plated for 24 h in full medium, then cultured for 6 additional hours in EBM-2 basal medium supplemented (FGF) or not (-FGF/NT) with 3nM FGF-2 as indicated. Upper panel: immunoblot for cleaved-caspase 3. Tubulin was used as a loading control. Lower panel: semi-quantification of cleaved-caspase 3 signal relative to tubulin signals versus the values of the NT group by ImageJ (n=3, unpaired t test, ***p<0.001). b, d Immunoblots for SRSF1, SRSF3, and SRPK1 (b) or phospho-SR (d) proteins in HUVEC treated (FGF) or not (NT) with 3nM FGF-2 for 6 or 24 h as in (a). GAPDH was used as a loading control. c, e Semi-quantification by ImageJ of the indicated proteins signals relative to GAPDH signal. The ratio obtained for the NT group was arbitrarily assigned the value 1 (c, n=4; e, n=3; unpaired t test, *p<0.05, **p<0.01, ns: not significant). f Left and middle panels: representative immunoblots of SRSF1 and SRSF3 protein levels (left) or RT-qPCR analyses of Srsf1 and Srsf3 mRNA levels (middle) in HUVEC transfected during 48 h with either control (Mis), SRSF1 (Srsf1), or SRSF3 (Srsf3) siRNA as indicated. A 50:50 mixture of two distinct SRSF1 or SRSF3 siRNAs was used (n=4; unpaired t test, ***p<0.001, ns: not significant). Right panel: cell viability quantified by using trypan blue exclusion counting in HUVEC just before plating for xCELLigence assay. Mean ± SD are presented (siRNA group mismatch, n=3; siRNA group Srsf1, n=2; siRNA group Srsf3, n=3). g xCELLigence assay on HUVEC transfected with the indicated siRNA and treated or not with 3nM FGF-2 (n=3)