Inhibitory profile of MLN518 against KIT mutant cell lines. (A) KIT juxtamembrane and activation loop mutant-expressing cell lines were assessed for cell viability by MTT assay at 72 hours in the presence of escalating doses of MLN518. Bcr-Abl Ba/F3 and Ba/F3 parental cell lines were used as controls. Graphs represent the average of 3 independent experiments, each plated in triplicate. (B) KIT mutant cell lines incubated in escalating doses of MLN518 were immunoblotted for tyrosine phosphorylation of the KIT receptor. Simultaneous blots for total KIT receptor were used as loading controls. Phosphotyrosine content was normalized for total KIT, and IC₅₀ values were calculated as the average of 3 independent experiments. Representative gels are shown. (C) KIT mutant cell lines were assessed for an increase in the number of apoptotic cells in response to escalating doses of MLN518. Apoptotic cell numbers were determined by flow cytometric measurement of annexin V binding. Representative experiments are shown. (D) KIT mutant cell lines were assessed for inhibition of phosphorylation of the downstream target Stat3. Phosphospecific immunoblots for tyrosine 705 of Stat3 were performed on cells incubated in the presence of either 1 μM MLN518 or 1 μM imatinib. Simultaneous blots of total Stat3 were used as a loading control. Representative gels are shown.