Phosphorylation of MAPKp42/p44, Akt, CREB, and ATF-1 in Ba/F3-Flt3 cells stimulated with FL and/or CXCL12 and effects of selective pathway inhibitors on migration to FL and/or CXCL12. (A) IL-3–starved cells were incubated with rhFL (10 ng/mL) and/or rhCXCL12 (100 ng/mL) for 5 minutes. Lysates were subjected to Western analysis for phosphorylation of MAPKp42/p44, Akt, CREB, and ATF-1. (B) Cells were preincubated with dimethyl sulfoxide (DMSO), 50 μM PD98059, 50 μM LY294002, or 10 μM H89 for 1 hour, washed twice, and subjected to migration to 10 ng/mL rhFL (□), 100 ng/mL rhCXCL12 (▪), or the combination of FL plus CXCL12 (▨) for 4 hours. ▦ indicates background migration (0/0). Cell viability was evaluated by forward and side scatter analysis following migration. Data represent mean ± SEM of percentage migration in 1 of 6 experiments with similar results. † indicates synergistic effect of FL plus CXCL12; P < .0001. (C) Ba/F3-Flt3 cells were preincubated with 10 μM AG1296 for one hour, washed, and evaluated for migration to 10 ng/mL FL (□), 100 ng/mL CXCL12 (▪), or a combination of FL plus CXCL12 (▨) as described in panel B. ▦ indicates background migration (0/0). Data represent the mean ± SEM relative percentage migration compared with cells pretreated with DMSO and migrating to FL plus CXCL12 (expressed as 100%) from 2 experiments of triplicate counts. † indicates synergistic migration to FL plus CXCL12; P < .0001.
