Midostaurin does not inhibit proliferation or FLT3 phosphorylation in Ba/F3 cells expressing an ITD and the G697R point mutation. (A) The proliferation of Ba/F3 cells expressing FLT3-ITD and G697R point mutation is not inhibited by midostaurin even at high doses. Ba/F3 cells expressing FLT3-ITD plus G697R were incubated with increasing concentrations of midostaurin for 48 hours. The number of viable cells was then determined using a colorimetric assay. Results are expressed as a percentage of viable cells after 48 hours of growth (± SD) in the presence of DMSO without the drug. The experiment was repeated in the presence of IL-3. (B) Midostaurin does not inhibit phosphorylation of FLT3, STAT5, or ERK in Ba/F3 ITD cells with the G697R mutation. Whole cell lysates were prepared from cells incubated with various concentrations of midostaurin for 20 minutes, resolved by SDS-PAGE as before, and immunoblotted with the antibodies indicated.