FIG. 6.
Free ATP levels are decreased in HIF-1α-null cells by approximately half at hypoxia. Following seeding at high density (5 × 105) and plating overnight, cells cultured in DMEM–high-glucose medium supplemented with 25 mM HEPES were left at normoxia or transferred to a hypoxia chamber for 24 to 28 h. The levels of free ATP were estimated based on the ATP-dependent luciferase activity present in whole-cell extracts as described in Materials and Methods. To normalize for differences in cell number between wild-type and null cells, the molar levels of free ATP were corrected for the levels of protein (in micrograms) present in the same cell extracts prepared for the luciferase assay. This graph represents the results of the average of three independent assays (± the SEM) minus the highest and lowest outlying datum points for each genotype, as described in the Materials and Methods (n = 4 per treatment/genotype). At normoxia (solid bars), the molar ATP levels per microgram of protein tended to be more variable in null cells than in wild-type cells, but there was no statistical significance in ATP production between either genotype. In contrast, at hypoxia (hatched bars), the levels of free ATP produced by null cells was approximately half of that produced by wild type cells and represented a statistically significant difference.