Figure 4.
Figure 4. Heme regulates both GATA1 mRNA and protein in erythroid cells. (A) GSEA enrichment plot of the hallmark heme metabolism pathway. Nominal P values for individual cells in clusters A-D of wild-type control, EPO-treated, and Flvcr1-deleted mice are shown. The hallmark heme metabolism pathway includes genes involved in both erythroid differentiation and heme synthesis. (B) Transcript expression levels (RPKM) of Gata1. 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-D) GSEA enrichment plots of genes in the GATA1 cluster (supplemental Table 5). Nominal P values for individual cells in clusters A-D of wild-type and Flvcr1-deleted mice (C) or cells in clusters A-D from wild-type, EPO-treated, and Flvcr1-deleted mice (D) are shown. GSEA is a nondeterminant method that calculates the reference baseline from all cells in the analysis. This reference baseline varies depending on the cells that are included. Inclusion of the cells from EPO-treated mice thus alters the GSEA reference baseline, which increases the nominal P value for many individual cells when panel D is compared with panel C. Cells from the Flvcr1-deleted mice show significantly less upregulation of GATA1 cluster genes than cells from either wild-type or EPO-treated mice. (E) Heat map showing changes in expression of GATA1 cluster genes. Log2 transformed relative expression levels of each of the 150 genes in the GATA1 cluster in individual cells grouped by cluster A-D isolated from either wild-type control or Flvcr1-deleted mice. (F) Studies in early (CD36+GlyA–) and intermediate-late (CD36+GlyA+) human erythroid cells treated with or without 0.5 mM ALA for up to 30 minutes. GATA1 protein and mRNA concurrently decrease within 15 minutes after adding ALA. Representative western blot images are below (RI, relative band intensity; in parenthesis are the values normalized to actin). Normalized expression levels are presented relative to untreated samples collected at the same time. Western blot band intensities were within the linear sensitivity range of the digital detection system. Additional representative blots are in supplemental Figure 12. This time course is informative, as these normal human erythroid marrow cells express FLVCR and will export excess heme induced by ALA treatment, effectively restoring homeostasis without affecting cell viability. Because of this, we limited these studies to 30 minutes or less. Data are presented as mean values ± SEM of 3 to 6 independent experiments. Differences relative to untreated (T0) were evaluated by 1-way ANOVA with the post hoc Tukey’s test. *(mRNA) P ≤ .05; ** or ††(protein) P ≤ .01; †††P ≤ .001.

Heme regulates both GATA1 mRNA and protein in erythroid cells. (A) GSEA enrichment plot of the hallmark heme metabolism pathway. Nominal P values for individual cells in clusters A-D of wild-type control, EPO-treated, and Flvcr1-deleted mice are shown. The hallmark heme metabolism pathway includes genes involved in both erythroid differentiation and heme synthesis. (B) Transcript expression levels (RPKM) of Gata1. 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-D) GSEA enrichment plots of genes in the GATA1 cluster (supplemental Table 5). Nominal P values for individual cells in clusters A-D of wild-type and Flvcr1-deleted mice (C) or cells in clusters A-D from wild-type, EPO-treated, and Flvcr1-deleted mice (D) are shown. GSEA is a nondeterminant method that calculates the reference baseline from all cells in the analysis. This reference baseline varies depending on the cells that are included. Inclusion of the cells from EPO-treated mice thus alters the GSEA reference baseline, which increases the nominal P value for many individual cells when panel D is compared with panel C. Cells from the Flvcr1-deleted mice show significantly less upregulation of GATA1 cluster genes than cells from either wild-type or EPO-treated mice. (E) Heat map showing changes in expression of GATA1 cluster genes. Log2 transformed relative expression levels of each of the 150 genes in the GATA1 cluster in individual cells grouped by cluster A-D isolated from either wild-type control or Flvcr1-deleted mice. (F) Studies in early (CD36+GlyA–) and intermediate-late (CD36+GlyA+) human erythroid cells treated with or without 0.5 mM ALA for up to 30 minutes. GATA1 protein and mRNA concurrently decrease within 15 minutes after adding ALA. Representative western blot images are below (RI, relative band intensity; in parenthesis are the values normalized to actin). Normalized expression levels are presented relative to untreated samples collected at the same time. Western blot band intensities were within the linear sensitivity range of the digital detection system. Additional representative blots are in supplemental Figure 12. This time course is informative, as these normal human erythroid marrow cells express FLVCR and will export excess heme induced by ALA treatment, effectively restoring homeostasis without affecting cell viability. Because of this, we limited these studies to 30 minutes or less. Data are presented as mean values ± SEM of 3 to 6 independent experiments. Differences relative to untreated (T0) were evaluated by 1-way ANOVA with the post hoc Tukey’s test. *(mRNA) P ≤ .05; ** or ††(protein) P ≤ .01; †††P ≤ .001.

Close Modal
This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal