Inhibition of TGF-β signaling increases erythroid development in RPS14-deficient models. Human BMCD34⁺ cells were transduced with lentivirus carrying shRNAs against RPS14 or Luc control. (A) After 5 days of transduction, cells were sorted for GFP+ and were cultured for an additional 2 days. Culture media were collected for a TGF-β ELISA. (B) After 5 days of transduction, cells were sorted for GFP+. Protein was collected and analyzed by using western blot analysis. β-Actin was used as a loading control. (C) After 3 days of transduction, cells were treated by SB431542 at the indicated concentration for 4 days and analyzed for the CD71 expression by using flow cytometry. (D) After 1 day of transduction, cells were treated by 2 μM of SB431542 for 4 days and sorted for GFP+. A total of 4000 cells of GFP+ cells were plated in methylcellulose media specific for CFU-E colonies and cultured for 1 week. Colonies were counted by an investigator blinded to the conditions. (E) Wild-type (WT) embryos stained with o-dianisidine showed strong brown signal on the yolk sac, indicating normal hemoglobin levels. rps14^−/− mutant embryos (MT) stained with o-dianisidine showed weak brown signal on the yolk sac, indicating reduced hemoglobin levels. MT treated with 2 concentrations of SB431542 showed improved staining signal of hemoglobin compared with DMSO-treated rps14^−/− MT. All embryos are ventral views. (F) Human BMCD34⁺ cells were transduced with lentivirus carrying shRNAs against RPS14 and ALK5. After 7 days of transduction, cells were analyzed for the CD71 expression by using flow cytometry. Data are representative of 3 independent transduction experiments. *P < .05, **P < .01, ***P < .001.