Figure 3. The efficient mTORC1 inhibitor Torin1 causes a dose-dependent increase in Mtb growth in co-infected hMDMs at low MOI.

(a) Representative immunoblots from two independent experiments showing the dose-response of Torin1 and rapamycin on the autophagy markers LC3B and SQSTM1 (p62) and phosphorylation of the mTORC1 downstream targets S6 and 4EBP1, with their respective β-actin loading controls, after 6 h treatment. Full length of the cropped blots are shown in Supplementary Fig. S3. (b) hMDMs were infected at the indicated MOI for 2 h, and then treated with/without 250 nM Torin1 for 3 days. The signal from luciferase expressing H37Rv was quantified, and the graph shows the ratio in total bacteria (lysate + supernatants) for Torin1 treated vs. untreated. Data are mean ± SEM from 6 independent experiments. *p < 0.05 using paired Student t-test. (c) hMDMs were pre-infected with/without HIV for seven days before infected with H37Ra (MOI = 0.2) for 2 hours. hMDMs were then incubated with/without rapamycin or Torin1 at increasing concentrations for 3 days. Graphs show the level of intracellular bacteria in cell lysates compared to day 0. Data are mean ± SEM from triplicate, representative of two independent experiments.