Fig. 3. Expression of markers and PDGF-B/PDGFRß in ESC-derived VEGFR2⁺ cells treated with various growth factors. (A) Expression of PECAM1, SMA, PDGF-B and PDGFRß was determined by quantitative RT-PCR; VEGFR2⁺ cells treated with VEGF-A (V), VEGF-A and FGF-2 (V/F), FGF-2 (F) or PDGF-BB (P) for 1.5 days. Vertical axes show relative expression. All expression data were normalized to GAPDH. Results were classified into two groups according to the pattern of expression. Expression of SMA and PDGFRß in FGF-2-treated (F; blue bars) and PDGF-BB-treated (P; red bars) cells, and that of PECAM1 and PDGF-B in VEGF-treated cells (V and V/F; green and blue/green bars, respectively). FGF-2- and PDGF-BB-treated cells (labelled F and P, respectively) in the two images on the left, and VEGF-A- and VEGF-A–FGF-2-treated cells (labelled V and V/F, respectively) in the two images on the right are shown as black bars because expression levels of corresponding genes were not markedly induced. (B,C) Expression of PDGF-B (B) and PDGFRß (C) was immunohistochemically determined in cells treated for 1.5 days. Distribution of SMA is shown for comparison in the lower panels. While PDGF-BB is expressed more in endothelium than in mural cells in the V/F treatment (red arrow), PDGFRß was present in SMA⁺ cells (yellow arrows) as well as in PECAM1^–/SMA^– cells (yellow arrowheads), but not in PECAM1⁺ cells. Bright-field images have been merged in all pictures to demonstrate the presence of endothelial sheets. Scale bars: 20 µm (B) and 50 µm (C).