Resistance of KIT D816V+ MCs to TNF. (A-C) ROSAKIT WT (green) and ROSAKIT D816V (red) cells were treated with different TNF concentrations and cell growth was evaluated by 3H-thymidine incorporation assay. ROSAKITWT cells were additionally incubated with infliximab (10 μg/mL) (B) or etanercept (10 μg/mL) (C). (D) ROSAKIT WT and ROSAKIT D816V cells were incubated with recombinant TNF (100 ng/mL) for up to 300 minutes as indicated. Whole-cell protein extracts were analyzed for BIM expression by immunoblotting. (E) ROSAKIT WT and ROSAKIT D816V cells were incubated with or without recombinant TNF (100 ng/mL) for 6 hours. Whole-cell protein extracts were analyzed for BIRC5 (survivin) protein expression by immunoblotting. Actin served as loading control for immunoblotting. A representative blot is shown in panels D and E. (F) ROSAKIT D816V cells were transduced with doxycycline-inducible RNAi targeting BIRC5 or an NTC. After TNF treatment (100 ng/mL) of doxycycline induced and noninduced cells, cell growth was assessed by 3H-thymidine incorporation assay. Results in panels A-C and F represent mean and standard error of the mean of 3 independent experiments. (G) Serial sections of paraffin-embedded BM from a patient with SM were immunohistochemically stained with an anti-tryptase antibody (left) or an anti-BIRC5 antibody (right). ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001.
