G6PD activity is required for heme detoxification in macrophages. (A) BMDMs were treated with 20 µM heme and/or 100 µM DHEA, and intracellular heme concentration was measured at 1, 3, and 6 hours (n = 4). (B) BMDMs were treated with 20 µM heme for 4 hours to create heme-loaded macrophages. Subsequently, heme-loaded BMDMs were washed and treated with vehicle or 100 µM DHEA, and intracellular heme was measured at 5, 6, 8, 10, and 16 hours (n = 4). (C) The difference in intracellular heme at 4 and 16 hours (representing 12 hours of degradation) was used to calculate the heme degradation rate in heme-loaded macrophages with or without the addition of DHEA (n = 4). (D) Schematic representing the requirement of G6PD activity for macrophage heme degradation. Data are represented as mean ± SEM. Statistical significance between 2 groups was determined by Welch’s unpaired t test. Significance between more than 2 groups was determined by one-way ANOVA with post hoc Tukey’s tests to determine differences between specific groups. *P < .05; **P < .01; ***P < .001. See also supplemental Figure 3.