Time course of hypoxia's effect on TGF-β mRNA levels in HUVECs.
(A) RNase protection analysis of HUVEC mRNA is shown. Total RNA was extracted from HUVECs after exposure to 20% O2 or 1% O2 at indicated times; 0 hours represents the normoxic sample. Each lane contained 10 μg total RNA hybridized to an antisense RNA probe cocktail that contained the templates for genes, protected fragments of which were separated on a 5% DNA sequencing gel and are indicated by arrows. (B) The graph represents the quantification results from 4 independent experiments. TGF-β2 signal was normalized to that from L32 (mRNA for ribosomal protein subunit); 0 hours is the mean of mRNA levels from control normoxic HUVECs. Each subsequent bar represents mRNA levels compared with its own normoxic control (not shown) at indicated hours. *P < .05. (C) TGF-β protein in supernatants from hypoxic and nonhypoxic HUVECs cultured for 48 hours was determined before and after heat activation to quantitate bioactive and total TGF-β levels, respectively, by MLEC bioassay. Results are from 4 independent experiments.
