TSC1 deficiency increases p53 activity and miR-34a expression in mast cells. (A) Increased mTORC1 activation in TSC1KO BMMCs. Cell lysates from IL-3 sufficient or depleted cultures for 10 hours were analyzed by immunoblot with the indicated antibodies. (B) Decreased mTORC2 and Akt activities in TSC1KO BMMCs. Akt Ser473 and Foxo3a Ser318/321 phosphorylation was detected as in panel A. (C) Increased p53 phosphorylation and accumulation in TSC1KO BMMCs. (D) Expression of miR-34a. Total RNAs from IL-3 sufficient or depleted for 6 hours were isolated and miR-34a expression was determined by qRT-PCR after reverse transcription. (E) Overexpression of miR-34a promotes mast cell death. WT BMMCs were transduced with lentivirus encoding GFP plus primary miR-34a. After 72 hours, the cells were grown in IL-3–depleted media for 24 hours, and then stained with annexin V and 7AAD. Data shown are mean ± SEM of annexin V–positive apoptotic cells from multiple samples. (F) Effect of miR-34a neutralization on death of TSC1KO BMMCs. TSC1KO BMMCs were infected with lentivirus encoding GFP plus sponge for miR-34s (SPNG-34s). After 24 hours culture in IL-3 depleted media, the cells were stained, analyzed, and graphically presented of annexin V–positive apoptotic cells from multiple samples (mean ± SEM; *P < .05; ***P < .001).