Fig. 2

Autophagy was increased upon inhibition of the mTOR-dependent autphagic pathway in wild type macrophages as expected, but not in TSC1 KO macrophages. a BMMϕ were treated with the mTOR inhibitor, rapamycin at 0, 10, 100 ng/ml for 24 h, and then lysed for Western blot analysis. b TSC1 KO BMMϕ were treated with autophagy inhibitors, 5 mM of 3-methyladenine, 1 μM of wortmannin, and 100 μM of bafilomycin A1 for 2 h. c Autophagic puncta were visulized using immunofluorescent microscopy (1,000X magnification). TSC1 WT and KO BMMϕ were grown in chamber slides and treated with rapamycin at 20 ng/ml overnight. d The number of puncta was counted in at least 20 cells for each group under the fluorescent microscope and average number of puncta per cells was calculated. e TSC1 KO BMMϕ were transfected with shLUC (control) or shRAPTOR lentivirus and selected in the presence of puromycin at 2 μg/ml for 4 days and Western blot analysis was performed with indicated antibodies including p-AMPKα (T172). All the experiments were repeated at least three times. ImageJ was used to quantify band intensities and the ratio of LC3B-II/Actin (loading control) is shown. The arrow indicates LC3B-II. Rapa, rapamycin; Un, uninfected; WT, Wild type; KO, knockout; ShLUC, shRNA control; ShRAPT, ShRAPTOR; 3-MA, 3-methyladenine; Wort, wortmannin; BafA1, bafilomycin A1; **, p < 0.01, ****, p < 0.0001