Figure 6
From: Hypoxia upregulates expression of human endosialin gene via hypoxia-inducible factor 2

Expression of endosialin in response to high cell density. (A) 42-MG-BA cells were plated to form sparse (S, 12 000 cells cm−2) and dense (D, 48 000 cells cm−2) monolayers and incubated for 24 h in normoxia. Gentle stirring was applied to dense cells to eliminate pericellular hypoxia. Total RNA was extracted and expression of endosialin mRNA was analysed by quantitative RT–PCR and normalised to β-actin levels. Stirring reduced the density-elevated expression of endosialin. (B) Endosialin promoter activity in sparse vs dense HeLa cells was assessed by transfection of −1091/+43, −235/+43 and −48/+43 fragments in pGL3-basic vector (1 μg) with pRL-TK plasmid (50 ng) as described in Figure 2. Effect of density was not observed in the shortest promoter construct. (C) Sparse HeLa cells were co-transfected with −235/+43 promoter fragment in pGL3-basic (1 μg), pRL-TK plasmid (50 ng) and SP1 or AP-2 expression vectors (1 μg each). Empty pCMV plasmid was used to adjust total DNA content in the control sample. The promoter activity was assessed after 24 h incubation in normoxia as described in Figure 2. SP1 but not AP-2 induced endosialin promoter activity. (D) Immunoblotting analysis of endosialin expression in sparse (S) and dense (D) 42-MG-BA cells treated with increasing concentrations of the SP1 inhibitor mithramycin A (MMA). M78 MAb revealed increased expression of endosialin in the dense cells and progressively reduced levels in MMA-treated cells. α-Tubulin antibody was used for a loading control. (E) Additive effect of high cell density and hypoxia on endosialin promoter activity was evaluated in HeLa cells transfected with the −1091/+43 promoter fragment as described in Figure 2A.