Figure 3.
IRX1 expression induces megakaryocytic and erythroid differentiation in ML-DS cells. (A) Growth of KPAM1 cells stably expressing either WT or mutant IRX1 (E321fs). KPAM1 cells were transduced with the IRX1-ZsGreen bicistronic retrovirus. Cell fate was monitored using flow cytometry. Ratios of ZsGreen-positive cells were determined on the days after infection with the viral transgenes. The combined results from 3 independent experiments are shown. Data are presented as mean ± standard deviation (SD). ∗P < .05; ∗∗P < .01 by Welch t test. (B) Western blot analysis of IRX1 expression. The analyzed cells included WT KPAM1 (lane 1), KPAM1-expressing WT IRX1 under the control of a Dox-inducible promoter before (lane 2), and after (lane 3) Dox treatment, and TAM (lanes 4, 5, and 6). The red arrow indicates IRX1 expression. (C) Cell cycle analysis of IRX1-inducible KPAM1 cells. The combined results from 3 independent experiments are shown. WT IRX1 induction led to an increase in the G0/G1-phase population and a decrease in the S-phase population. Data are presented as mean ± SD; ∗P < .05, Welch t test. (D) RNA-seq analysis of KPAM1 cells. Gene set enrichment analysis (GSEA) of transcriptomes in WT IRX1-inducible KPAM1 cells after vs before IRX1 induction. (E) GSEA using a gene set of MYC targets, showing that MYC was significantly downregulated upon WT IRX1 expression. The red arrow indicates MYC expression. (F) GSEA using a gene set for megakaryocytic (left) and erythroid (right) differentiation. (G) Flow cytometric analysis of IRX1-inducible KPAM1 and MGS cells. KPAM1 and MGS cells were treated with Dox for 6 days. Representative data are shown for 1 of 4 independent experiments. (H) Morphology of IRX1-inducible KPAM1 and MGS cells before and after WT IRX1 induction for 7 days. Cytospin specimens were stained with May-Grünwald Giemsa (original magnification ×10 000). (I) Cell pellets from KPAM1 cells before and after WT IRX1 induction. KPAM1 cells were treated with Dox for 7 days. (J) Zebrafish orthologs of human IRX1, irx1a, and irx1b (GenBank accession numbers: NM_207184 and NM_ 131823) were knocked down using morpholino antisense oligos (MOs) that targeted the translational initiation sites of each gene. One-cell–stage embryos were injected with these MOs, and the density of blood cells around the cardiac vein at 48 hours after fertilization was detected by hemoglobin staining using o-dianisidine. Embryos injected with irx1a MO and/or irx1b MO displayed anemia (severe, moderate, or mild anemia) depending on the concentration of the injected MOs (1, 5, and 10 μg/μL). Black arrows indicate hemoglobin-stained blood cells (orange dots) in wild-type embryos. Black and gray arrowheads indicate embryos with severe and moderate anemia, respectively. The number of embryos analyzed for hemoglobin staining is shown at the top of the bars. FDR, false discovery rate; NES, normalized enrichment score; n.s., not significant.

IRX1 expression induces megakaryocytic and erythroid differentiation in ML-DS cells. (A) Growth of KPAM1 cells stably expressing either WT or mutant IRX1 (E321fs). KPAM1 cells were transduced with the IRX1-ZsGreen bicistronic retrovirus. Cell fate was monitored using flow cytometry. Ratios of ZsGreen-positive cells were determined on the days after infection with the viral transgenes. The combined results from 3 independent experiments are shown. Data are presented as mean ± standard deviation (SD). ∗P < .05; ∗∗P < .01 by Welch t test. (B) Western blot analysis of IRX1 expression. The analyzed cells included WT KPAM1 (lane 1), KPAM1-expressing WT IRX1 under the control of a Dox-inducible promoter before (lane 2), and after (lane 3) Dox treatment, and TAM (lanes 4, 5, and 6). The red arrow indicates IRX1 expression. (C) Cell cycle analysis of IRX1-inducible KPAM1 cells. The combined results from 3 independent experiments are shown. WT IRX1 induction led to an increase in the G0/G1-phase population and a decrease in the S-phase population. Data are presented as mean ± SD; ∗P < .05, Welch t test. (D) RNA-seq analysis of KPAM1 cells. Gene set enrichment analysis (GSEA) of transcriptomes in WT IRX1-inducible KPAM1 cells after vs before IRX1 induction. (E) GSEA using a gene set of MYC targets, showing that MYC was significantly downregulated upon WT IRX1 expression. The red arrow indicates MYC expression. (F) GSEA using a gene set for megakaryocytic (left) and erythroid (right) differentiation. (G) Flow cytometric analysis of IRX1-inducible KPAM1 and MGS cells. KPAM1 and MGS cells were treated with Dox for 6 days. Representative data are shown for 1 of 4 independent experiments. (H) Morphology of IRX1-inducible KPAM1 and MGS cells before and after WT IRX1 induction for 7 days. Cytospin specimens were stained with May-Grünwald Giemsa (original magnification ×10 000). (I) Cell pellets from KPAM1 cells before and after WT IRX1 induction. KPAM1 cells were treated with Dox for 7 days. (J) Zebrafish orthologs of human IRX1, irx1a, and irx1b (GenBank accession numbers: NM_207184 and NM_ 131823) were knocked down using morpholino antisense oligos (MOs) that targeted the translational initiation sites of each gene. One-cell–stage embryos were injected with these MOs, and the density of blood cells around the cardiac vein at 48 hours after fertilization was detected by hemoglobin staining using o-dianisidine. Embryos injected with irx1a MO and/or irx1b MO displayed anemia (severe, moderate, or mild anemia) depending on the concentration of the injected MOs (1, 5, and 10 μg/μL). Black arrows indicate hemoglobin-stained blood cells (orange dots) in wild-type embryos. Black and gray arrowheads indicate embryos with severe and moderate anemia, respectively. The number of embryos analyzed for hemoglobin staining is shown at the top of the bars. FDR, false discovery rate; NES, normalized enrichment score; n.s., not significant.

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