Fig. 6.

Knockdown of the m⁶A MTase complex blocks erythropoiesis in human adult, bone marrow-derived HSPCs. a Flow cytometry of WTAP KD, METTL3-KD, and METTL14-KD hBM HSPCs differentiated in liquid culture reveals a block to erythropoiesis with no impact on megakaryopoiesis and an enhancement of myelopoiesis with METTL3-KD (n = 3, mean ± SEM, t-test two-sided). b Flow cytometry of erythroid progenitor populations reveals an early block to erythropoiesis following WTAP KD. At day 6, WTAP KD CD235+/CD71+ cell numbers were only 8.4% of scr control and by day 10, WTAP KD CD235+/CD71− cell numbers decreased further to 5.5% of control cells. c Colony formation assays from WTAP KD hBM HSPCs confirm the block to erythropoiesis (n = 6, mean ± s.d., erythroid t-test two-sided, p < 0.0000001). d MeRIP-qPCR validation of m⁶A methylation for key erythroid regulators and genes translationally decreased following WTAP KO in a bulk population of adult BM erythroid cells (ERY1, ERY2, and ERY3) pooled from six donors. (n = 1). e Flow cytometry of lv-shRNA-KD of the translationally altered genes identified in Fig. 4c in hBM HSPCs differentiated in erythroid promoting liquid culture reveals a delay or reduction in erythropoiesis at day 8. (n = 2, mean). f Flow cytometry for differentiation potential of hBM CD34 + HSPCs transduced with lv-shRNA of the translationally altered genes identified in Fig. 4c and cultured in megakaryocyte or myeloid liquid culture differentiation conditions shows no effect on those lineages, except for enhanced myelopoiesis following CXXC1 KD and BRD7 KD (n = 2, mean ± range, t-test two-sided). g Single-cell western blot analysis of WTAP KD MEPs recapitulates the reduction in PABPC1 and PABPC4 protein observed in WTAP KO HEL cells (data from a minimum of 250 cells were used per sample, Kolmogorov–Smirnov)