FIG. 1.

Calcium ionophore A23187 does not induce HIF-1-dependent transcription in MEF or in A549 cells. (A) HIF-1-dependent transcription was not affected by the calcium ionophore in A549 cells. Cells were transfected with 1 μg of HRE-Luc or 1 μg of plasmid CMV-Luc by using TransFast transfection reagent. On the day after transfection, cells were treated in some instances with 0.16 or 0.8 mM nickel chloride, hypoxia (1% O₂), or 5 μM A23187 for 20 h. Cells were lysed and luciferase activity was determined as described previously (28). The data are the ratios of luciferase expression on HRE-Luc to that on CMV-Luc (HRE/CMV Ratio). (B) HIF-1-dependent transcription was not affected by calcium ionophore in MEF. The exposure conditions were similar to those described for panel A. (C) The HIF-1α protein level was not affected by calcium ionophore. Sixty micrograms of nuclear extracts from A549 cells untreated or treated with 5 μM A23187, 10 μM BAPTA-AM, or 260 μM DFX for 20 h was resolved by SDS-8% PAGE and subjected to Western blot analysis for detection of HIF-1α and p53 as described in Materials and Methods. The detection of another nuclear protein, p53, in the same samples was conducted to confirm protein loading in the cells.