Expression of EPOR and FLI1 discriminates the progressive commitment of progenitor cells in the GPA⁺CD41⁺ cell fraction. (A) Scatter plot indicating transcript levels for KLF1, EPOR, FLI1, and MPL in Ery, MEP, and MK fractions. Each dot represents the absolute number of transcripts present in each cell. Horizontal bars represent the average levels for each gene in the various populations calculated considering the log normal distribution of the data (Shapiro-Wilk normality test run to confirm that the transcript distribution is log-normal at 95% significance level; P = .05). The absolute number of transcripts for each gene was retrieved by comparing threshold cycle values of the cDNAs to those obtained from a dilution series of a well-defined quantity of a purified PCR product. (B) Unsupervised cluster analysis (K-means) of expression of EPOR, MPL, KLF1, and FLI1 in Ery, MEP, and MK fractions at a single-cell level. The gene transcript values of the 4 genes in the overall fractions were integrated together to assess for independent clustering in specific patter of expression. Three clusters were identified: cluster Ery, cluster MEP, and cluster MK (top panel). Names were given according to the main cell type represented in each cluster (cluster Ery with 52% of cells GPA⁺CD41⁻, cluster MK with 64% of cells GPA⁻CD41⁺). The cluster MEP was equally represented by the GPA⁺CD41⁻, GPA⁺CD41⁺, and GPA⁻CD41⁺ cell subsets. Profiles of gene expression were derived to describe each individual cluster (bottom panel). (C) Dot plot distribution between EPOR and FLI1 at a single-cell level. Spots from Ery, MEP, and MK clusters were colored differently. Each spot represents a single cell. Here, 3 clouds of points are observed, where cells in the MEP cluster progressively commit to the Ery or MK cluster by increasing the number of copies of EPOR or FLI1 transcripts, respectively.