MITF knockdown inhibits cell growth in c-KIT mutant cells. (A) Western blot shows efficient knockdown of MITF protein expression with sh-RNA-expressing lentivirus (si-MITF) in murine (P815) and human (HMC-1) mastocytoma lines. Control lentivirus targeting β-actin (si-β-actin) shows no effect on MITF expression. (B) MITF repression results in decrease in cell proliferation. BrdU incorporation of both murine and human mastocytoma cells with si-MITF–expressing lentivirus is decreased approximately 40% of control. *P < .5. **P < .005. (C) MITF repression significantly impairs colony-forming capacity of mastocytoma lines. Representative images of mastocytoma colonies transduced with lentiviral empty vector, si-β-actin, and si-MITF in methylcellulose. Murine (P815) and human (HMC-1.1) cells with knockdown of MITF (si-MITF) are impaired in colony-forming capacity. Lentiviral vector expresses green fluorescent protein, and green fluorescent protein–expressing cells are visualized (original magnification ×10). Fluorescent images were obtained with a Nikon fluorescent microscope with excitation filter set at 488 nm. Images were cropped with Photoshop Elements 2.0 (Adobe Systems). (D) MITF resistant to silencing (MITF rescued) restores colony-forming capacity. For controls for potential off-target effects of sh-RNA and lentivirus, an MITF-expressing retrovirus with and without the si-RNA target site was constructed. Western blot shows human mastocytoma cells (HMC-1.1) expressing MITF with si-RNA binding site mutated (MITF rescued) is resistant to silencing, but the wild-type MITF-expressing cells (MITF) are not. (E) Human mastocytoma cells (HMC-1.1) are dependent on MITF expression for colony formation. Knockdown of MITF with the si-MITF-expressing lentivirus impairs colony formation MITF-expressing cells (vector and MITF, white bars), but cells with MITF resistant to silencing (MITF rescued) maintain colony-forming capacity. **P < .005. (F) Constitutive tyrosine phosphorylation of mutant D814V KIT. 293T cells were stably transduced with vector, wild-type KIT, and mutant D814V retrovirus and then immunoprecipitated with an anti-KIT antibody. Western blots performed for phosphotyrosine and KIT expression. Both wild-type and D814V KIT-transduced cells show expression of KIT, but only D814V KIT-transduced cells show significant constitutive tyrosine phosphorylation. Total input from immunoprecipitated lysates was probed for α-tubulin expression and showed roughly equivalent starting material. (G) D814V requires MITF expression for mast cell growth. Cell proliferation of BMMCs in response to SCF (72 hours incubation) was measured with XTT assay (measured at OD 450 nm). Wild-type BMMCs (white bars) proliferate in response to SCF; MITF^−/− BMMCs (black bars) do not proliferate as well to high doses of SCF. MITF^−/− BMMCs transduced with vector (horizontal bars), KIT (gray bars), or mutant D814V KIT (diagonal bars) retrovirus do not restore proliferative capacity of MITF^−/− cells. **P < .005. P values are calculated with Student t test; all experiments performed in triplicate.