Fig 2: SF3A2 is a Key regulator of Human Erythropoiesis and Modulates Erythropoiesis Defects in a Murine Model of MDS.(A) Quantitative RT-PCR and (B) Western blot showing the expression of SF3A2 in human CD34+ cells five days post-infection with the respective lentiviral shRNAs targeting SF3A2 (sh1-4) and a control luciferase gene (shLUC). (C) Growth curves showing that downregulation of SF3A2 results in reduced total cell numbers during erythroid differentiation from three independent experiments. (D) Representative FACS plots of erythroid cell surface markers CD71 (transferrin receptor) and CD235a (Glycophorin A) expression at various time points during erythroid differentiation. Percentages in each quadrant are represented as mean and standard deviation of three independent experiments (E) Altered splicing events identified by RNA-Seq analysis of stage matched erythroid cells (shSF3A2 vs. shLUC). Overlapping changes observed in SF3B1 mutant BM cells from MDS patients (Obeng et al) (Figure 6—source data 5 and 6). Differentially expressed genes and pathway analysis available in Figure 6—source data 1–4. (F) Lineage negative bone marrow cells from wildtype (WT) and Sf3b1K700E mice were infected with shRNAs targeting murine Sf3a2 gene co-expressing a reporter GFP gene. Percentage of Ter119+ CD71+ erythroid cells within the GFP compartment after 48 hr in erythroid differentiation. (G) Total cell numbers of GFP+ erythroid cells after 48 hr in erythroid differentiation.10.7554/eLife.44080.022Figure 6—source data 1.Table containing the DESeq2 output for differentially expressed genes in cells undergoing SF3A2 knockdown or control shRNA treatment.10.7554/eLife.44080.023Figure 6—source data 2.Table containing the DESeq2 output for differentially expressed genes in MDS patients with and without mutations in SF3B1.10.7554/eLife.44080.024Figure 6—source data 3.Tables containing the GO component (Table 1) and function (Table 2) enrichments calculated using GOrilla for cells undergoing SF3A2 knockdown or control shRNA treatment.10.7554/eLife.44080.025Figure 6—source data 4.Tables containing the GO component (Table 1) and function (Table 2) enrichments calculated using GOrilla for MDS patient samples with and without mutations in SF3B1.10.7554/eLife.44080.026Figure 6—source data 5.Tables containing the differential splicing analysis for cells undergoing SF3A2 knockdown or control shRNA treatment.Categories of splice mutations presented in each table are alternative 3’ splice sites, alternative 5’ splice sites, mutually exclusive exons, retrained introns, and skipped exons, respectively.10.7554/eLife.44080.027Figure 6—source data 6.Tables containing the differential splicing analysis for MDS patient patient samples with and without mutations in SF3B1.Categories of splice mutations presented in each table are alternative 3’ splice sites, alternative 5’ splice sites, mutually exclusive exons, retrained introns, and skipped exons, respectively.