Hypoxia increases MIF mRNA expression (A) and protein production (B, C) in HUASMCs. Growth-arrested HUASMCs were exposed to hypoxia for different durations. (A) Quantitative RT-PCR (Q-PCR) results. Total cellular RNA was isolated from normoxia or hypoxia-stimulated cells. After reverse transcription, they were subjected to quantitative PCR analysis to determine MIF mRNA level. Graph is representative of relative MIF mRNA levels in the various conditions (n = 3 in each group). * indicates P < 0.05 vs control cells under normoxia. (B) Release of MIF protein measured by ELISA. Growth-arrested cells were exposed to hypoxia (3% O2) for different durations, and MIF protein released into cell culture media was measured by ELISA (n = 3 in each group). * indicates P < 0.05 vs control cells under normoxia. (C) Expression of MIF protein detected by Western blot. Western Blot detecting of MIF expression in control HUASMCs under normoxia or hypoxia (3% O2) for different durations. Representative Western blot (top) and values of MIF production (mean ± SEM of 3 experiments, bottom). Results of MIF protein production were obtained from densitometric analysis and expressed as ratio of MIF/β-actin. * P < 0.05 vs control cells under normoxia.