Effect of the MEK inhibitor U0126 on the growth of BL-CFC and progenitors.

(A) EBs3 cells derived from mpl^−/− flmpl or mpl^−/− Δ3mpl ES cells were pretreated with 10 or 20 μM U0126 or 20 μM DMSO for 1 hour and then stimulated with VEGF, PEG-rhuMGDF, or VEGF+PEG-rhuMGDF for 15 minutes. Whole-cell extracts were then subjected to SDS-PAGE and Western blot analysis with phospho-specific anti-ERK1/2 or anti-ERK1/2 antibody. Images were made with Adobe Photoshop 5.0. (B) EBs3 or EBs6 cells were pretreated with various concentrations of U0126 or DMSO for 1 hour and then replated for blast cell colony assay (left panel) or progenitor assay (right panel) with PEG-rhuMGDF alone. (C) EBs3 derived from mpl^−/− flmpl ES cells were treated as described above and replated for blast cell colony assay with PEG-rhuMGDF, VEGF, or VEGF+PEG-rhuMGDF (left panel). Ratios of the numbers of blast cell colonies stimulated with VEGF+ PEG-rhuMGDF to those stimulated with VEGF were calculated (right panel). (MGDF: PEGrhu MGDF).