Loss of TSC1 increases mast cell apoptosis. (A) Increase in size of TSC1KO BMMCs determined by FACS analysis. Mean ± SEM of forward cell scatter (FCS-A) of 3 samples are shown. (B) Increased death of TSC1KO BMMCs. WT and TSC1KO BMMCs were cultured in the presence or absence of IL-3 for the indicated time. Viable cells were counted by trypan blue exclusion. Data shown are mean ± SEM from 3 paired samples. (C) Increased apoptosis of TSC1KO BMMCs. BMMCs were stained with annexin V and 7AAD after 48 hours IL-3 depletion. (D) mTORC1 signaling induced by SCF stimulation. BMMCs were rested for 1 hour in Tyrode buffer before stimulation with SCF (30 ng/mL). Phosphorylation of p70S6K, 4E-BP1, and Akt were assessed by immunoblot. Blots were stripped and probed for total protein control. (E) Increased death of TSC1KO BMMCs cultured in SCF. Cells were cultured at the indicated conditions for 30 hours and stained with annexin V. (F) Mast cell proliferation. CFSE-labeled WT and TSC1KO BMMCs were cultured in the presence of IL-3, SCF, or SCF ± IL-3 for 72 hours followed by FACS analysis. (G) Mast cell numbers in the spleen, lung, and skin. Mast cells in the spleen, lung, dorsal skin, and ears were stained with toluidine blue and were counted under a light microscope. WT and TSC1^f/f-ERCre⁺ mice were injected with tamoxifen on days 1, 2, and 5 and were euthanized for harvesting spleen, lung, dorsal skin, and ear on day 14. Data shown are mean ± SEM of 4 to 7 mice. Top panels, representative toluidine blue staining of spleen thin sections. Bar graphs represent mean ± SEM of mast cell numbers per high power field (400×) from multiple mice. Data shown are representative of at least 3 experiments (**P < .01; ***P < .001).