Gene expression analysis using GeneCall technology and RT-PCR. (A) Primary HUVECs were exposed for 0, 6, or 24 hours to either serum-free media or serum-free media complemented with VEGF or 5% FCS, respectively. Endothelial cells were lysed and total RNA was isolated and analyzed for gene expression by using GeneCalling technology as described previously.³⁶ Each column represents the output of a series of binary comparisons that have been compared to each other. However, genes marked with “P” (P = poisoning) or “I” (I = Isolation, cloning, sequencing, and poisoning) were confirmed. Selected genes shown were found induced by VEGF at 6 and 24 hours, but not by stimulation by 5% FCS; the fold stimulation by VEGF is indicated by a number in each box. Gray boxes indicate that no change in expression was observed for the genes in the binary comparisons for serum at 6 and 24 hours versus no serum. Vertical black bars in the enlarged thumbnail indicate the confidence level of the gene call. (B) DSCR1 expression analysis by real-time PCR.²¹  HUVECs were kept in serum-free conditions and stimulated with either 5% FCS, b-FGF (10 ng/mL), VEGF (30 ng/mL), PlGF (100 ng/mL), VEGFR1-sel (100 ng/mL), or VEGFR2-sel (30 ng/mL) during the indicated amount of time. The mutant proteins used were VEGFR2-sel (KDR-selective): VEGF D63S/G65M/L66R and Flt-selective VEGF I43A/I46A/Q79A/I83.²⁵  (C) DSCR1 expression analysis by real-time PCR. HPAECs, HDMECs, and HUAECs were incubated in presence of 0.5% FCS (NA), or in presence of 0.5% FCS and VEGF (30 ng/mL) or VEGF mutant forms, binding to either VEGFR1 (VEGFR1-sel, 100 ng/mL) or VEGFR2 (VEGFR2-sel, 30 ng/mL)²⁶  for 4 (▪), 8 (▦) , and 24 (□) hours. As control cells, HUPASMCs were used, which do not express significant levels of VEGF receptors (data not shown). Data shown represent the average ± SD of triplicate samples of one representative of total 3 independent experiments. Relative RNA units (RRUs) are calculated as described by Gerber et al³  by using cyclophylin as reference gene. (D) DSCR1 expression analysis by real-time PCR. HUVECs were incubated for 2 hours in presence of CsA (1 μM) prior stimulation with VEGF (30 ng/mL), VEGFR2-sel (30 ng/mL), VEGFR1-sel (100 ng/mL), PMA (200 ng/mL), and IO (5 μM), thapsigargin (50 nM) or TNF-α (10 ng/mL) for 5.5 hours, as indicated. Data shown represent the average ± SD of duplicate samples run in parallel of one representative of total 2 independent experiments. (E) Western blot analysis of whole cell extracts of HUVECs grown in 0% serum, 0.5% BSA and 5% FCS, hVEGF (30 ng/mL), b-FGF (10 ng/mL), TNF-α (10 ng/mL) and PMA or ionomycine (IO) for 5 hours as indicated. As control, 100 ng recombinant DSCR1 protein was included.