Pharmacologic inhibition of KIT signaling results in MITF protein reduction. (A) D816V mutation confers resistance to imatinib treatment. XTT assay shows sensitivity of HMC-1.1 (KIT D816V-negative) and relative resistance of HMC-1.2 (KIT D816V-positive) to imatinib. (B) D816V-negative and -positive HMC cell lines are sensitive to midostaurin. XTT assays were performed in triplicate and absorbance measured at OD 450. Relative OD 450 value is calculated by normalizing OD 450 reading to OD 450 with no treatment. (C) MITF protein expression is reduced with inhibition of KIT signaling. Western blot shows that both phosphotyrosine signal and MITF protein are reduced with imatinib treatment in HMC-1.1. In HMC-1.2, phosphotyrosine signal is only mildly reduced and MITF protein is not significantly repressed. (D) Midostaurin treatment results in inhibition of phosphotyrosine in both HMC-1.1 and HMC-1.2. Western blot for α-tubulin shows equivalent loading. Relative expression is calculated by taking the ratio of the densitometry signal for MITF to tubulin and normalizing to 1.0 for the zero time point.