Ectopic expression of KITD816V in wild-type and TET2-deficient mast cells. (A) Lentiviral infection of in vitro differentiated BMMCs with human KITD816V. (B) Tet2 wild-type (+/+), heterozygous (+/−), and homozygous (−/−) BMMCs were infected with LV-KITD816V at day 0 and GFP expression to detect infected cells over time. n = 6 biologic replicates. (C) Cells were pulse-labeled with BrdU for 24 hours. Quantitation of percent BrdU-positive cells is shown for both GFP/KITD816V-positive and -negative cells. Independent replicates are graphed as points; data are mean ± SEM for each dataset on the graph. Pairwise comparison of datasets was performed using the Mann-Whitney test, and P values are indicated where differences were significant. (D) Cell viability was assessed by trypan blue exclusion and counted using a hemocytometer for Tet2 wild-type (+/+), heterozygous (+/−), and homozygous (−/−) BMMCs infected with KITD816V over time after withdrawal of IL-3 from culture medium. n = 4 biologic replicates. (E) Annexin V staining for samples as in panel D 0-48 hours in the absence of IL-3. (F) Percent GFP-positive cells within total live cell fraction from (D) at day 6 after IL-3 withdrawal. (G) Fluorescence profile of Cell Trace Violet at days 0, 3, 6, and 10 in the presence or absence of IL-3 in labeled BMMCs. Representative profiles are shown for each genotype. Error bars represent SEM of biologic replicates.