Figure 4: YTHDC1 contributes to human HSPC myeloid differentiation.

(A-I) Human CB-CD34+ cells were transduced with lentiviruses expressing control shRNA or two independent shRNAs targeting YTHDC1. Cells were used for following experiments after puromycin selection. n=3 independent experiments. (A) Immunoblot of YTHDC1 expression in CB-CD34+ cells. (B) Cell proliferation of control and YTHDC1 depleted CB-CD34+ cells were determined. (C) Colony forming assay of control and YTHDC1 depleted HSPCs. The total number of colony forming units (CFUs) was scored two weeks after plating. (D) Apoptotic cells were determined by flow cytometry at day four and six post-transduction. (E-G) Myeloid differentiation of CB-CD34+ cells was measured by flow cytometry using CD11b, CD13, CD14 and CD33 as markers at indicated timepoint. Representative flow plot was shown in (E). (H and I) Erythroid differentiation of CB-CD34+ cells was measured by flow cytometry using CD71 and glycophorin A(GYPA) as markers at indicated timepoint. (J-L) CB-CD34+ cells were transduced with lentiviruses expressing control, YTHDC1 and its different mutants as indicated. Sorted cells were used for following experiments. n=3 independent experiments. (J) Representative immunoblot of YTHDC1 expression in indicated CB-CD34+ cells. (K) Cell proliferation of CB-CD34+ cells were determined. (L) Cells in (K) were plated on methylcellulose (5000 cells for each replicate). The total number of colony forming units (CFUs) was scored two weeks after plating.

Error bars, s.e.m. * p<0.05, **p<0.01, ***p<0.001, two-tailed t test.