SelenoW is a candidate regulator of erythroid maturation. (A) Protein expression analysis by western blot of SelenoW in sorted spleen CD71⁺cells, ProEs and BasoEs from Se-D and Se-A mice on day 3 after 50% PHZ treated; n = 2-3. (B) ENCODE RNA-Seq analysis of SelenoW in G1E and G1E-ER4 cells after GATA-1 activation by E2 treatment. (C-E) Analysis of SelenoW knockdown or control G1E and G1E-ER4 cells after E2 treatment. (C) Percentage of Ter119⁺ cells (n = 3) and (D) percentage of neutral benzidine⁺ cells (n = 6) generated 48 and 72 hours after treatment with E2 in G1E and G1E-ER4 cells transduced with SelenoW gRNA or a nontargeting control gRNA. (E) Analysis of cell cycle in SelenoW mutant or control G1E-ER4 cells. Percentage of G₂/M phase cells in the culture; n = 3. (F-H) SelenoW knockdown in TgCas9-GFP BM cells. SelenoW gRNA was expressed from mCherry-expressing lentivirus. GFP⁺mCherry⁺ cells are transduced cells. GFP single-positive (GFP⁺) cells are nontransduced cells. (F) Fold increase in the percentage of GFP⁺mCherry⁺ cells transduced with SelenoW gRNA (gRNA) or nontargeting control gRNA (NTC) after 7 days in SEEM expansion culture and after switching the cultures to 3-day differentiation culture (SEDM day 3 after SEEM). Two independent repeats. (G) Analysis of stress BFU-E colonies generated in the 2-phase culture. Cells were plated after 3 days in SEDM media. Cell were plated in methylcellulose media containing only Epo at 20% O₂ or supplemented with BMP4 and stem cell factor at 2% O₂. Two independent repeats. (H) Stress BFU-E colony assays of sorted GFP⁺mCherry⁺ or GFP⁺ cells from SelenoW gRNA transduced cultures after 2-phase culture. Cells were plated in methylcellulose media containing only Epo at 20% O₂; n = 4 per type of cells. Pairwise Student t test was used for statistical analysis. (I) Colony assays of human BM cells after 2-stage stress erythroid culture. Frozen human bone marrow cells from 3 subjects were thawed 24 hours before transduction with huSelenoW dCas9-KRAB/sgRNA lentivector or dCas9-KRAB empty lentivector control. The cells were transduced by spinoculation, and left in culture with virus overnight. On the second day, cells were transferred to SEEM and cultured for 7 days, followed by 3-day SEDM culture. Transduced (GFP⁺) cells were purified by sorting and plated for colonies. Bars are representative of mean ± SEM. *P < .05; **P < .001; **P < .005; ****P < .0001.