Mek1,2 inhibition reverses EpoR-HM erythroblast stage-specific differentiation defects. (A) Bone marrow–derived wt-EpoR, EpoR-HM, and EpoR-H erythroid progenitor cells were cultured for 72 hours in SP34-EX medium containing U0126 (± 10 μM). Expanded erythroblasts then were differentiated (in transferrin, BSA, and insulin-containing medium) with Epo at 2.5 U/mL and U0126 (± 10 μM). At 40 hours of culture, frequencies of high forward-angle light scatter erythroblasts were assayed. Note the reversal of differentiation defects in EpoR-HM erythroblasts as illustrated by U0126-induced decreases in forward scatter (cell size). (B) U0126 reversal of EpoR-HM erythroblast differentiation defects as analyzed by Ter119 and CD71 marker expression. At 40 hours of differentiation, EpoR-HM erythroblasts also exhibited significantly decreased frequencies of Ter119^pos erythroblasts specifically within a subpopulation of maturing cells with decreased CD71 expression. U0126 reversed this defect (but had no significant effects on control wt-EpoR cells). (C) U0126 dose-dependent reversal of EpoR-HM erythroblast differentiation defects also was observed based on U0126-dependent increases in frequencies of Ter119^pos erythroblasts. (D) U0126 inhibition of ERK1,2 activation in primary wt-EpoR, EpoR-HM, and EpoR-H erythroblasts. The capacity of U0126 to inhibit the Epo-stimulated activation of ERKs was confirmed directly by exposing expanded, Ter119-depleted erythroblast preparations to ± 10 μM U0126. In scatterplots, the numbers in quadrants (clockwise from bottom left) indicate the percentage of CD71^negTer119^neg, Ter119^negCD71^pos, CD71^posTer119^high, and Ter119^highCD71^neg cells among total live-gated cells. Boxes in each top right quadrant indicate the percentage of CD71^highTer119^high (top) and CD71^lowTer119^high (bottom) populations.