Modulation of HIF-1-dependent PAI-1 promoter and EPO-HRE luciferase activity by A23187 and BAPTA-am. HepG2 cells were transfected with either luciferase (Luc) gene constructs driven by a wild-type 796-base pair (bp) human PAI-1 promoter (pGL3-hPAI-796), or the 796-bp promoter mutated at the HRE-2 (pGL3-hPAI-796-M2) site. In addition, Luc gene constructs containing 3 copies of the EPO HRE element in front of the simian virus 40 (SV40) promoter (pGl3EPO-HRE) or mutated EPO-HREm were used. The transfected cells were treated with A23187 (0.1 μM), BAPTA-am (5 μM), or BAPTA (5 μM) and further cultured 24 hours under normoxia (16% O2) or hypoxia (8% O2). In each experiment the LUC activity of pGL3-hPAI-796, pGL3-hPAI-796-M2, pGL3-EPO-HRE, or pGL3-EPO-HREm transfected cells at 16% O2 was set to 100%. Values are means ± SEM of 3 independent culture experiments. Statistics, Student t test for paired values: *P ≤ .05, versus 16% O2 in the same group, **P ≤ .05, versus the control groups at the same pO2.
