Figure 1.

TMEM187 is an erythroid differentiation suppressor in vitro. (A-B) Relative mRNA levels of TMEM187 in multiple cell lines (A) and of genes encoding HBA, HBB, HBG, ALAS2, and FECH during induction of K562 cells (B), detected by quantitative RT-PCR. Erythroid differentiation of K562 cells was induced by glutamine deprivation, the same as in later experiments. n = 3/group. (C) Protein levels of erythroid differentiation-related genes in K562 cells, detected by immunoblotting. (D-E) Dynamics of TMEM187 mRNA (D) and protein levels (E) during induction of K562 cells, detected by qPCR (n = 6/group) and immunoblotting, respectively. The color of cell pellets (panel E middle) and in-gel heme staining (panel E bottom, blue bands) are shown. (F) Confirmation of TMEM187 KD or KO in K562 cells by western blotting. Ponceau S stain was used as an internal control. (G-H) Cell pellets of K562 KD (G) and KO (H) mutants prior to and after induction. (I) HBA expression in K562 KD and KO mutants, detected by immunoblotting. (J-K) O-dianisidine staining of hemoglobin in K562 KD and KO cells. GAPDH or ACTB was used as an internal control for immunoblotting unless otherwise specified, and 18S for qPCR. t test for panel A and 1-way ANOVA for panel D were used for significance. ∗∗∗P < .001; ∗∗∗∗P < .0001. ACTB, actin beta; ANOVA, analysis of variance; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; RT-PCR, reverse transcription polymerase chain reaction.