Cbl mutants synergize with Kit to induce autonomous growth and in vitro colony formation. (A-B) Cbl-70Z and Cbl-R420Q induce ligand-independent proliferation and survival of 32D-Kit-WT cells. The 32D cells stably overexpressing Kit-WT and/or indicated Cbl mutants were starved from IL-3, and cells were grown in the presence of 10% FCS. Cells were counted at the indicated time points by the trypan blue exclusion method, and the data are shown as fold change of the cell number compared with the start of the experiment (A). (B) The percentage of cells in the culture that was alive at the indicated time points. (C) Ligand-independent DNA synthesis of Kit-WT cells coexpressing Cbl-70Z. The 32D cells stably expressing Kit-WT and/or Cbl constructs were starved from IL-3 for 12 hours and then cultured in the presence or absence of SCF or IL-3. Proliferation was measured by ³[H]-thymidine incorporation assays. Data are shown as percentage of thymidine incorporation relative to the thymidine incorporation of the respective cell line with IL-3 supplementation. (A-C) Data represent the average and SD of 3 independent experiments. (D-E) Cbl mutants led to cytokine-independent colony growth. The 32D cells stably overexpressing Kit-WT and/or indicated Cbl mutants were serum starved for 12 hours and then plated at a concentration of 1000 cells per dish in the absence of any growth factors. Colonies were counted on day 6. The assays were plated as triplicates. The numbers given show the results of 1 of at least 3 independent experiments per construct, which all gave similar results. (E) Photographs of the dishes were taken on day 6 and show morphologic differences in clonal growth.