Fig. 8
FGF-2 mediates accumulation of sVEGFR1 splice variants through a SRSF1/SRSF3/SRPK1-dependent axis. a HDMEC were transfected with control siRNA (mis) or specific SRSF1 (Srsf1) or SRSF3 (Srsf3) siRNA and treated with 3nM FGF-2 for 72 h. Left panel: representative immunoblots of SRSF1 or SRSF3 protein (WB) or PCR analyses (RT-PCR) of sVEGFR1-i13 and sVEGFR1-ex12 mRNA levels. GAPDH was used as a loading/internal control. Right panel: PCR data semi-quantification by ImageJ software. The sVEGFR1/GAPDH ratio obtained in control condition was arbitrarily assigned the value 1. Data are the mean ± SD of 3 and 4 biological replicates for sVEGFR1-ex12 and sVEGFR1-i13, respectively (unpaired t test, **p<0.01, ***p<0.001, ns: not significant). b sVEGFR1-i13 and sVEGFR1-ex12 mRNA levels were quantified by RT-qPCR in HUVEC transfected with either control siRNA (mis) or specific SRSF1 or SRSF3 siRNA and treated with 3nM FGF-2 for 48 h. GAPDH was used as an internal control. Mean ± SD are presented (n=3 biological replicates, unpaired t test, ***p<0.001, ****p<0.0001, ns: not significant). c, d VEGFR1 (R1), sVEGFR1-ex15 (R1-Ex15), sVEGFR1-i13 (R1-i13), and sVEGFR1-ex12 (R1-Ex12) mRNA levels were quantified by RT-qPCR in HUVEC (c) or HDMEC (d) treated (FGF, white bars) or not (NT, black bars) with 3nM FGF-2 for 48 h (HUVEC) or 72 h (HDMEC) in the presence or absence of 10nM AZD4547 (FGFRinh, gray bars), 10μM SRPIN340 (SRPIN340, black hatched bars), or 5μM SPHINX31 (SPHINX31, gray hatched bars). GAPDH was used as an internal control. Mean ± SD are presented (n=6; 2 technical replicates of 3 independent experiments, unpaired t test, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns: not significant). e Quantification by ELISA assay of sVEGFR1 protein level in HDMEC supernatants collected 1 day or 3 days after treatment with or without (control) 3nM FGF-2 and 5μM SPHINX31 or 10μM SRPIN340. Data represent the mean ± SD of 3 (day 2) or 5 (day 4) independent experiments. t test, *p< 0.05, **p< 0.01, ***p<0.001, and **** p<0.0001