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Figure 6 mTORC1 inhibition cannot ameliorate RagCS75Y cardiomyopathy both in vitro and in vivo. (A,B) Immunoblot of P-p70S6K, P-S6, TFEB, and α-SA in NRVCMs. The NRVCMs were infected with Ad:GFP (CON) or Ad:RagCS75Y (S75Y) for 24 h followed by rapamycin (100 nmol/L) or torin (10 nmol/L) or vehicle incubation for another 24 h. (C) Neither rapamycin nor torin rescue the enlarged cell size induced by RagCS75Y. Data were averaged from three independent experiments. * p < 0.05; NS, not significant. Data are mean ± SEM by one-way ANOVA. (D), mtor haploinsufficiency (mtor+/−) did not rescue reduced cardiac function in rragc KI fish. Shown are echocardiographic analysis of EF and FS in the WT control, single mutants, and rragc KI;mtor+/− double mutants at 7 months. n = 8,11,8,10. (E) The abnormal elevated vmhc mRNA expression was not attenuated in rragc KI;mtor+/− double mutant fish at 7 months compared with rragc KI fish. n = 3,5,5,5. Data in (D,E) are shown in boxplot (MIN to MAX). * p < 0.05, NS, not significant versus rragc KI, one-way ANOVA. (F), Kaplan–Meier survival curves of rragc KI;mtor+/− double mutant fish compared with their corresponding single mutants and the WT control by log-rank test. (G). Rapamycin marginally attenuated the increased phosphorylation of TFEB at S211 in S75Y cells. Quantification data was shown as mean ± SEM, * p < 0.05, one-way ANOVA. (H). Rapamycin did not promote TFEB nucleus translocation in S75Y cells. Shown are representative immunoblots of TFEB, RagC, and P-S6 proteins in the nuclear and cytosolic fractions of H9C2 cardiomyocytes infected with Ad:GFP or Ad:RagCS75Y and then treated with 100 nmol/L Rapamycin (S75Y + Rap) or vehicle for 24 h. Lamin A/C and LDH were used as nuclear and cytosolic protein loading control, respectively. (I) AD293 cells were transfected with TFEB–FLAG and RagCWT-HA or RagCS75Y-HA; 24 h later, cells were treated with vehicle or rapamycin (100 nmol/L) or vehicle for another 24 h. Cells were lysed and subjected to immunoprecipitation with the anti-HA antibody. The immunoprecipitates and cell lysates were analyzed by immunoblotting with antibodies against HA (used to detect RagC) and FLAG (used to detect TFEB). IP: immunoprecipitation; IB: immunoblot. Quantification data mean ± SEM, * p < 0.05, one-way ANOVA.