Lipid rafts are required for proliferation induced by wild-type and oncogenic Kit. (A) Effect of MβCD on KL-mediated proliferation of Mo7e cells. ³H-thymidine incorporation over a time period of 12 hours of Mo7e cells stimulated with KL for 24 hours in the absence or in the presence of imatinib (1 μM) or MβCD at the indicated concentrations was determined. The stimulation index was measured as the ratio of incorporated radioactivity by KL-stimulated cells and unstimulated cells, measured in triplicate (all values ± 1 SD). (B) Reversibility of the growth inhibitory effect of MβCD on Mo7e cells. To rule out irreversible toxic effects of MβCD, a washout experiment was performed for the incubation of cells in the presence of 4 mM MβCD, the highest concentration used in A. The stimulation index was determined using Mo7e cells incubated in either the absence or presence of 4 mM MβCD. After 24 hours, cells were washed and resuspended in fresh media with or without MβCD for 6 hours before ³H-thymidine incorporation in the presence or absence of KL for 12 hours. (C) Effect of MβCD on the proliferation of HMC-1 cells. ³H-thymidine incorporation over a time period of 12 hours was analyzed after incubation of the cells in media alone or in the presence of either imatinib (1 μM) or MβCD for 24 hours at the indicated concentrations. Relative proliferation refers to the ratio of incorporated radioactivity by cells at their respective media conditions and untreated cells. (D) Comparison of the effect of imatinib on murine Kit^558V>G and Kit^814D>V expressed in Ba/F3 cells. Cells were incubated for 12 hours in either media alone or media containing 1 μM imatinib. Immunoprecipitated Kit protein was analyzed for tyrosine phosphorylation (top) and the presence of Kit protein (bottom). (E) Analysis of the effects of imatinib and MβCD on Akt activation mediated by Kit^558V>G or Kit^814D>V. Lysates from Ba/F3 Kit^558V>G or Ba/F3 Kit^D814V cells incubated for 6 hours in media alone or in the presence of 1 μM imatinib or MβCD at the concentrations indicated were separated by SDS/PAGE and analyzed with antibodies against phosphotyrosine, Kit, and phosphorylated Akt (Ser⁴⁷³). (F) Growth inhibitory effect of MβCD on Ba/F3 Kit^558V>G or Ba/F3 Kit^814D>V cells. ³H-thymidine incorporation of Ba/F3 Kit^558V>G or Ba/F3 Kit^814D>V cells was analyzed in the absence or in the presence of imatinib (1 μM) or MβCD at the indicated concentrations. Relative proliferation refers to the ratio of ³H-thymidine incorporation of cells in their respective media conditions and in media alone.