Fig 2: SF3B1 regulates ELK1 mRNA splicing.(A) Flow cytometry analysis of neutrophils and monocytes derived from L-ELK1-overexpressing CD115- CD34+ progenitors. Flow cytometry analysis was performed on day 9.(B). Identification of different ELK1 isoforms in MoPs and NbPs by PCR. Ten nanograms of dsDNA generated from MoPs or neutrophil-biased progenitors (NbPs) was used for PCR.(C) Immunofluorescence for ELK1 N-terminus and ELK1 C terminus antibodies were performed to assess L-ELK1 and S-ELK1 expression levels in MoPs and neutrophil biased progenitor (NbP2, NbP3) cells. For the absence of C-terminus domain of S-ELK1, the C-terminus antibody indicates L-ELK1 only and the ELK1 N-terminus antibody indicates both L-ELK1 and S-ELK1.(D) GSEA was performed to analyze the effect of ALA on RNA splicing-related signal changes in NbP1.(E) Heatmaps present ALA-upregulated and ALA-downregulated RNA splicing-related genes.(F) Flow diagram for mass spectrometry (MS) analysis of ELK1 RNA pull-down proteins.(G) GO enrichment analysis of ELK1 RNA pull-down proteins. Proteins with coverage >10% (391 proteins) were subjected to GO enrichment analysis.(H) Scatter plots showing the relationship of peptide-spectral matches (PSM) and protein coverage. Proteins with coverage >30% and PSM >20 are denoted by red gene names.(I) Statistical analysis of the proportions of neutrophils and monocytes derived from SRSF1 shRNA- and SF3B1 shRNA-transduced CD371+ CD115- GMPs. Flow cytometry analysis was performed on day 14 of differentiation.(J) RNA pull-down and immunoblotting were performed to measure the interaction of ELK1 RNA and SF3B1 under ALA treatment.(K) Immunofluorescence staining for ELK1 N-terminus and ELK1 C- terminus antibodies were performed to assess L-ELK1 and S-ELK1 expression levels in SF3B1-shRNA transduced NbP1 cells and Ctrl NbP1 cells. For the absence of C-terminus domain of S-ELK1, the C- terminus antibody indicates L-ELK1 only and the ELK1 N-terminus antibody indicates L-ELK1 and S-ELK1 Data in (A, I) are represented as the means ± SD. An unpaired Student's t-test (two-tailed) was performed to determine statistical significance. NS = not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, N = 3–7. (C, J) Scale bar = 20 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig 3: ALA regulates commitment of neutrophil progenitors by targeting ELK1.(A) Statistical analysis of neutrophils and monocytes differentiated from NbP1s, NbP2s, and NbP3s ± ALA treatment. To analyze the effect of ALA on neutrophil differentiation, sorted neutrophil biased progenitors were cultured ± ALA and flow cytometry assay to performed to analyze the CD11b+CD15+CD66b+CD14– neutrophils and CD11b+ CD66b–CD14+ monocytes at day6 to day14.(B) Volcano plots and GO enrichment analysis of genes upregulated and downregulated by ALA.(C) Heatmaps showing ALA-downregulated genes among neutrophil-biased progenitor-specific genes.(D) Heatmaps presenting ALA-downregulated transcription factors in NbP1s and NbP2s.(E) Flow cytometry analysis of neutrophils and monocytes derived from CD34+ HSPCs transduced with shRNAs targeting ALA-downregulated transcription factors. The ratio of neutrophils to monocytes is presented to analyze the neutrophil differentiation potential of CD34+ HSPCs transduced with different shRNAs.(F) Flow cytometry plots of neutrophils and monocytes from ELK1-shRNA knockdown CD34+ progenitors. Flow cytometry analysis was performed on day 9, and neutrophils and monocytes were analyzed with CD11b+ gated cells. Representative flow cytometry plots and statistical analysis bar plots are shown.(G) Immunofluorescence analysis of ELK1 was performed to measure ELK1 protein levels in NbP1 cells ± ALA treatment. Scale bar = 20 μm.(H–I)In vitro flow cytometry analysis of neutrophils and monocytes from ELK1-shRNA-transduced NbP1, NbP2, and NbP3 cells. Flow cytometry analysis was performed from day 6 to day 9, and neutrophils and monocytes were analyzed within GFP+ CD11b+ gated cells. Representative flow cytometry plots (G) and statistical analysis bar plots (H) are shown.Data in (A, E, I) are expressed as means ± SD. An unpaired Student's t-test (two-tailed) was performed to assess statistical significance. NS = not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, N = 3–4.