KIT D816V induces expression of CCL2. (A) Mo7e and TF-1 cells were transduced with the empty vector (CO), wild-type (WT) KIT, or KIT D816V. Then, expression of total and phosphorylated KIT (pKIT) were determined by immunoblotting. Actin served as a loading control. (B) Mo7e and TF-1 cells were transduced as described and cultured in the absence or presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) for 16 hours. Green fluorescent protein (GFP) –positive cells were purified by fluorescence-activated cell sorting (FACS), and expression of cytokines was determined by quantitative real-time PCR. Results are expressed as ΔCt values (ΔCt = Ct_ABL − Ct_cytokine). Samples without detectable mRNA expression were set to a ΔCt value of −10.0. Data from 4 experiments are shown. (C,D) TF-1 cells were transduced and cultured as described above. Expression of CCL2 was determined by (C) real-time PCR of cells or (D) ELISA of cell culture supernatants. (E) Cord blood–derived MCs harvested at different time points and KIT D816V⁺ HMC-1 cells were analyzed for expression of CCL2. (F) HMC-1 cells were treated with midostaurin for 16 hours, and expression of CCL2 was determined by real-time PCR. (G) Expression of CCL2 was analyzed in HMC-1 cells after transduction with a short hairpin RNA–targeting KIT (KIT RNAi) or a nontargeting control (NTC). Results represent the mean ± standard deviation (SD) of at least 3 independent experiments. **P < .01, ***P < .001.