Figure 2.

TMEM187 deficiency reprograms K562 cells in favor of erythropoiesis. Differentiation was induced by glutamine deprivation for 4 days unless otherwise specified. (A) Heme biogenesis proteins and hemoglobin subunits, detected by immunoblotting. GAPDH was used as an internal control. (B) mRNA levels of hemoglobin subunits, detected by qPCR. 18S was used as an internal control. One-way ANOVA was used for significance. n = 6/group. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001. (C) Representative UV-visible spectra of cell lysate (equal amounts of total proteins). The embedding graph is in-gel heme staining. (D-E) Cell membrane protein CD71 and CD235a analysis by flow cytometry and their quantification. One-way ANOVA was used for significance. n=3/group. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001. (F) Immunoblotting for band 3 in WT and KO cells after 7-day induction. Ponceau S stain was used as an internal control. Oligomer, polymerized band 3; full-length band 3, 102 kDa; C-terminal domain of band 3, 55 kDa; N-terminal domain of band 3, 40 kDa. (G-H) Representative fluorescence images (G) and quantification (H) of DAPI (4′,6-diamidino-2-phenylindole) staining (yellow, pseudocolored) for nuclear size assessment in WT and KO cells after 7-day induction. t test was used for significance. n = 60/group. ∗∗∗∗P < .0001. (I) Gene set enrichment analysis of hematopoiesis before (left) and after 4-day induction (right). (J) Cluster heat map of genes for hemoglobin synthesis and erythrocyte membrane proteins, derived from RNA-sequence. NES, normalized enrichment score.