Heme increases ribosomal protein content in early erythroid cells. (A) GSEA enrichment plot of the KEGG ribosome pathway. Nominal P values for individual cells in clusters A-D of wild-type control, EPO-treated, and Flvcr1-deleted mice are shown. (B) Transcript expression levels (RPKM) of ribosomal protein genes. Mean values ± SD for each cluster are graphed. Differences in gene expression levels between the cell clusters from wild-type control mice and Flvcr1-deleted or EPO-treated mice were identified by t test. (C-F) Studies in early human erythroid cells (CD36⁺GlyA^–) treated with or without 0.5 mM ALA for up to 30 minutes. ALA rapidly increases intracellular heme content (C), resulting in elevated ROS (D) and the increased expression of RPS19, RPS24, and RPS26 protein and mRNA (E). Representative western blots are in supplemental Figure 5. Levels of 18s and 28s rRNA also increase (F). The expression levels of experimental samples were normalized relative to the untreated control samples collected at the same time. Mean values ± SEM for 3 to 4 independent studies are shown. Differences relative to untreated (T0) were evaluated by 1-way ANOVA with a post hoc Tukey’s test. *(mRNA) or †(protein) P ≤ .05; ** or ††P ≤ .01; ***P ≤ .001.