Knockdown of ncoa4 impacts erythropoiesis in zebrafish and tissue culture cells.

(A, B) Expression of HGBG (A), HGBA (B) determined by quantitative RT-PCR in K562 cells expressing a control (shGFP) or two independent shRNAs targeting NCOA4. Hemin treatment (25 μM, 72 hr) as indicated. (C) Immunoblot analysis of K562 cells as in (A) and (B) with indicated antibodies. Hemin treatment (25 μM, 72 hr) as indicated. (D) Appearance of K562 pellets as in (A, B, and C). Red coloration indicates appropriate hemoglobinization of cells after hemin differentiation. Brown coloration indicates accumulation of iron without hemoglobinization. (E) Expression of ncoa4 in circulating erythroid cells relative to sites of primitive hematopoiesis. Abbreviations: hpf = hours post-fertilization, ICM = intermediate cell mass, YS = yolk sac, H = heart, L = liver. (F) Circulating RBCs are visualized in globin-LCR:eGFP erythrocyte reporter zebrafish at 30 hpf. Morpholino-mediated knockdown of ncoa4 severely disrupts erythropoiesis (n > 30 each condition). Inset shows erythrocytes circulating in the caudal artery and caudal vein plexus. (G) FACS quantification of erythrocytes in globin-LCR:eGFP reporter zebrafish at 30 hpf following control or ncoa4-morpholino-mediated knockdown (ncoa4 MOa and MOb). ***p < 0.001 and **p < 0.002 by two-tailed unpaired t-test. (H) o-dianisidine staining (brown) for hemoglobinized red cells in zebrafish embryos. Embryos were grown from zygotes injected at the one- to two-cell stage with ncoa4-targeting morpholinos (MOa and MOb) or control injected zygotes (control). Ncoa4 MOa: 15/16 embryos with diminished staining in comparison to control. Ncoa4 MOb: 18/22 embryos with diminished staining in comparison to control.