Receptor phosphorylation, IL-3–independent growth, and response to PKC412 of Ba/F3-FLT3-N841I cells. (A) Expression and phosphorylation of the FLT3-N841I receptor. Whole-cell extracts were immunoblotted with either anti-pTyr antibody to detect receptor phosphorylation or anti-FLT3 to detect the level of receptor expression (as indicated). Lines 1 to 3 represent 3 independent polyclonal cell populations stably expressing FLT3-N841I and maintained in media without IL-3. FLT3-ITD represents the Ba/F3 cells expressing FLT3-ITD (as a positive control). pClneo represents the Ba/F3 cells expressing empty vector (as a negative control). (B) FLT3-N841I induces the IL-3–independent growth of Ba/F3 cells. Ba/F3 cells stably expressing FLT3-N841I were seeded at a density of 0.5 × 105/mL and were grown in the presence or absence of IL-3 (as indicated). As a control, Ba/F3 cells containing only the empty vector (pClneo) and growing in the presence of IL-3 were also seeded at the same density. Cells were collected at the indicated times and counted after staining with trypan blue. (C-D) Dose-dependent growth inhibition of Ba/F3-FLT3-N841I and Ba/F3-FLT3-ITD cells by PKC412. Ba/F3-FLT3-N841I cells (C) and, as a control, Ba/F3-FLT3-ITD cells (D) were seeded at 0.5 × 105/mL and were immediately treated with PKC412 at the indicated concentrations. Cells were collected at 72 hours and counted as in panel B. All data points are the average of experimental duplicates and are representative of the results obtained in 2 independent experiments.
