Heme synthesis rapidly downregulates GATA1 and upregulates β-globin in K562 cells. (A) K562 cells were treated with or without 0.5 mM ALA or ALA and 0.5 mM succinylacetone and analyzed for heme content, ROS levels, GATA1 protein, and β-globin protein content. Normalized expression levels are presented relative to untreated samples collected at the same time. As expected, within 15 minutes after ALA addition, heme content, ROS and β-globin protein increased and GATA1 protein decreased. These changes fail to occur when ALA (which bypasses the first step in heme synthesis) and succinylacetone (which blocks the second step in heme synthesis) were both added, thus demonstrating that the outcomes are heme-dependent. (B) Representative western blots for GATA1 and β-globin protein quantitation (RI, relative band intensity; in parenthesis are the values normalized to actin) at 15 minutes. NT, no treatment. An additional representative blot is in supplemental Figure 16. (C) K562 cells transduced with a heme reporter construct containing wild-type β-globin enhancer and promoter elements driving luciferase or a negative control construct containing mutant MARE elements⁸  were treated with 0.5 mM ALA for up to 30 minutes and assayed for luciferase expression. Luciferase expression is a measure of regulatory heme, as it quantitates heme-dependent transcription. Relative luciferase levels are presented as fold over the negative control construct harvested at the same time. All data are presented as mean values ± SEM of 3 to 4 independent experiments. Differences relative to untreated (T0) samples were identified by t test analysis. *P ≤ .05; **P ≤ .01; ***P ≤ .001.