PCA reveals a distinct transcriptional program in cells from Flvcr1-deleted mice. (A) α-globin (Hba-a1, left) and β-globin (Hbb-bs, right) gene expression levels (RPKM) grouped by Ter119-based clusters (A-D) for each marrow sample. Cells from Flvcr1-deleted mice have significantly higher globin transcript levels than either control, showing that heme, but not EPO, increases globin transcription. Mean values ± SD are presented. 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. *P ≤ .05; **P ≤ .01; ***P ≤ .001. (B) Two-component PCA of all gene expression with the mean gene expression trajectory of each cell cluster A-D shown as individual vectors for cell clusters from wild-type mice (indicated WA-WD) and Flvcr1-deleted mice (indicated DA-DD). Group A cells from both mice (WA and DA) are transcriptionally similar, as shown by their colocalization; however, their subsequent differentiation follows different trajectories. The erythroid cells from wild-type mice (WB to WD) progress clockwise, whereas cells from Flvcr1-deleted mice (DB to DD) progress counterclockwise. As anticipated, the dying cluster A cells (DAd) are transcriptionally distinct. Erythroid differentiation genes were generally distributed along the y-axis, whereas proliferation genes were generally distributed along the x-axis. Some genes with large projections are identified. (C) An independent 2-component PCA of cell clusters from EPO-treated mice (indicated EA-ED) shows that the erythroid differentiation of these clusters tracks comparably to clusters from wild-type mice (WA-WD). The same genes are highlighted as in panel B to facilitate comparison. Together, the analyses show that erythroid cells from wild-type and EPO-treated mice are transcriptionally similar throughout differentiation, whereas the transcriptional profile of erythroid cells from Flvcr1-deleted mice differs from both controls.