Figure 5.

Constitutive MAPK pathway activation contributes to the KIT^D816V-induced block in differentiation. (a) Primary erythroblast cells were cytokine-starved for 4h and stimulated for 10 min with 1 μg/ml SCF or 10 units/ml Epo as indicated. Cells were then lysed to analyze activity of key signaling molecules by immunoblotting. Results shown are representatives of three independent experiments. (b–d) Erythroblast cells were stimulated to differentiate in the presence of MEK1/2 inhibitor U0126 (10 μM) or DMSO as solvent control. (b) Cells were staged according to morphology and hemoglobin accumulation. Vav:KIT^D816V: N=3. Controls: N=3. BasoE, basophilic erythroblast; OrthoE, orthochromatic erythroblast; PolyE, polychromatic erythroblast; Reti, reticulocyte. (c) Cells were analyzed by flow cytometry and staged according to their CD71/Ter119 expression profile. Graph shows quantification for day 3 of differentiation. Statistical analysis was performed by means of two-way ANOVA with Bonferroni post-tests. (d) Relative expression levels of indicated genes determined by qRT-PCR on day 2 of differentiation in KIT^D816V cells treated with U0126 and corresponding solvent controls (normalized to Gusb and Sdha). Vav:KIT^D816V: N=3. Controls: N=3; P-values were determined using one-way ANOVA with Bonferroni post-tests. Variance given as S.D. *P<0.05; **P<0.01; ***P<0.001