GPS2 promotes erythroid cell differentiation depending on EKLF. (A-D) Control cells and MEL cells with stable overexpression of WT GPS2 or GPS2 deletion mutants were left untreated or treated with HMBA for indicated times. Immunoblot analysis of GPS2 and EKLF protein levels (A). Hemoglobin production was analyzed by benzidine staining at days 3 and 4. Representative images of benzidine-stained cells are shown; scale bar, 20 μm (B). The percentage of benzidine-positive cells was calculated (C). Real-time PCR analysis of Ahsp and Hbb-b1 mRNA levels at day 3 (D). (E) CD34⁺GFP⁺ cells were selected from human UCB CD34⁺ cells infected with lentivirus expressing WT GPS2 or GPS2 deletion mutants and cultured in the erythroid differentiation medium for the indicated times. The proportion of CD71⁺GPA⁺ cells at days 0, 3, and 5 was analyzed by flow cytometry. (F-G) Human UCB CD34⁺ cells infected with NCOR1 or control shRNA lentivirus were cultured in the erythroid differentiation medium for the indicated times. The proportion of CD71⁺GPA⁺ cells at days 0, 3, and 5 was analyzed by flow cytometry (F). mRNA levels of the indicated erythroid genes in FACS-isolated CD71⁺GPA⁺ cells at day 3 were analyzed with real-time PCR (G). (H-L) Control cells and MEL cells with stable GPS2 knockdown or GPS2 knockdown and EKLF overexpression were left untreated or treated with HMBA for the indicated times. EKLF and GPS2 protein levels were analyzed by western blot (H). A representative photograph of the color of cell pellets at day 4 (I). Hemoglobin production was analyzed by benzidine staining at days 4 and 5. Representative images of benzidine-stained cells are shown; scale bar, 20 μm (J). The percentage of benzidine-positive cells was calculated (K). Relative mRNA levels of Alas2, Ahsp, and Hbb-b1 at day 3 were analyzed by real-time PCR (L). (M) CD34⁺GFP⁺RFP⁺ cells were selected from human UCB CD34⁺ cells infected with GPS2 shRNA lentivirus and EKLF overexpression or control lentivirus and then cultured in erythroid differentiation medium for the indicated times. The proportion of CD71⁺GPA⁺ cells at day 0, 3, and 5 was analyzed by flow cytometry. All values are mean ± SEM (n = 3 replicates). *P < .05, **P < .01, ***P < .001; 2-tailed unpaired t test.