Figure 1. m6A inhibits myeloid differentiation of human stem/progenitor cells (HSPCs).
(a–e) Human cord blood CD34+ (HSPCs) cells were transduced with lentiviruses expressing either a scramble (control) shRNA or two independent shRNAs targeting METTL3 (#9 and #12; METTL3-KD). (a) Cells were selected for puromycin resistance and immunoblotted at four days post-transduction. Left, representative immunoblot. Right, the quantitative summary n=3 independent experiments; error bars, s.e.m. **p<0.01,***p<0.001 two-tailed t test.
(b) m6A levels in poly(A) purified mRNA were quantified by two-dimensional thin layer chromatography (2D-TLC, see methods). n=3 independent experiments; error bars, s.e.m. * p<0.05, two-tailed t test.
(c) The number of viable cells was measured over the course of seven days beginning four days post-transduction of shRNAs. n=3 independent experiments; error bars, s.e.m. * p<0.05, two-tailed t test.
(d) The percentage of apoptotic cells was determined at day four and five post-transduction. Cells were stained for Annexin V and DAPI and quantified by flow cytometry.
(e) Myeloid differentiation was measured using CD11b and CD14 as markers of myeloid differentiation. Cells were stained and expression of each surface marker was quantified by flow cytometry seven days after plating. error bars, s.e.m. * p<0.05, **p<0.001, two-tailed t test.
(f–h) Human cord blood CD34+ (HSPCs) cells were transduced with retroviruses expressing GFP together with empty vector (EV) or wild type METTL3 or catalytically dead METTL3 (METTL3-CD). Cells were sorted based on GFP positivity two days post transduction.
(f) At XX time point cells were analyzed by XXX method. Immunoblots at two days post transductions n=3 independent experiments; error bars, s.e.m. ** p<0.01, two-tailed t test.
(g) Sorted cells were plated in basic media (See Supplementary methods). Cells were counted for seven days after plating. EV: Empty vector (black line), METTL3 (red line), catalytically dead METTL3-CD (gray line). n=4 independent experiments; error bars, s.e.m. * p<0.05, two-tailed t test.
(h) Myeloid differentiation was evaluated as in (e) seven days after plating in myeloid differentiation conditions. n=4 independent experiments; error bars, s.e.m. * p<0.05, *** p <0.0001 two-tailed t test.