Figure 5.

Effect of kinase inhibitors on survival of resting neutrophil progenitors and upon pro-inflammatory stimulation. (a) Wt progenitor cells were SCF withdrawn and treated for 6 h with various kinase inhibitors (LY294002 (20 μM), JI1 (1 μM), U0126 (10 μM)) as indicated in the presence or absence of LPS, GM-CSF or G-CSF. Cells were harvested and analyzed for apoptosis by intracellular staining for active caspase-3 followed by flow cytometry analysis. Data represent mean/S.E.M. of three independent experiments. *P≤0.05. (b) Wt progenitor cells were treated as described in a for 6 h. Cells were harvested, stained for loss of membrane integrity by propidium iodide and analyzed by flow cytometry. Data represent mean/S.E.M. of four independent experiments. *P≤0.05. (c) Wt progenitors were treated with the kinase inhibitors LY294002 (20 μM), JI1 (1 μM), U0126 (10 μM) or solvent control (DMSO, 0.2%) as indicated for 4 or 22 h. After harvest, cells were lysed and heated in Tricine sample buffer, and equivalents of 0.6 × 10⁶ cells were subjected to SDS-PAGE on 16% Tricine gels, transferred onto a nitrocellulose membrane and probed for A1 and Mcl-1. GAPDH served as loading control. Shown is one out of three independent experiments