Bafilomycin a1

Mouse adrenocortical Y1 and mouse progenitor Leydig TM-3 and Leydig MA-10 cell lines were grown in Dulbecco’s modified Eagle medium (DMEM)-F12 medium supplemented with 10% fetal bovine serum at 37 °C in a humidified atmosphere at 5% CO2. These cells were regularly examined for the presence of Ad4BP/SF-1and free of mycoplasma contamination by immunoblot, immunofluorescence, and DAPI staining according to the guidelines. HEK 293 cells were grown in DMEM supplemented with 10% fetal bovine serum and 1x penicillin-streptomycin-glutamine at 5% CO2 and 37 °C. For drug treatment, cells were incubated with or without 10, 50, or 100 μM chloroquine (Novus Biologicals, Littleton, CO, USA), 10 or 20 mM ammonium chloride (Sigma, St. Louis, MO, USA), 1, 5, or 10 nM bafilomycin A1(Enzo Life Sciences, Farmingdale, NY, USA), 2, 5, or 10 mM 3-methyladenine (Sigma, St. Louis, MO, USA), and 1 mM sodium pyruvate (Caisson Laboratories, Logan, UT, USA) for 16 or 24 h before analysis. 50 μg/ml cycloheximide(Sigma, St. Louis, MO, USA) for 2, 4, 6, or 8 h before analysis; 20 ng/ml platelet-derived growth factor alpha polypeptide (Cell Guidance Systems, Cambridge, UK) for 24 h before analysis.

HeLa cells were purchased from ATCC (line CCL‐2). The cells were cultured in full media, which consists of DMEM (01–052‐1A, Sartorius Israel), 10% heat‐inactivated bovine serum (FBS, 04‐007‐1A, Biological Industries) penicillin/ streptomycin (1000 μg/mL, 15240–062, Gibco Thermo Fisher Scientific), and L‐Glutamine (G7513‐100ML, MERCK). HeLa cells were cultured at 37°C in an atmosphere of 5% CO₂ and were routinely tested for mycoplasma. For autophagy induction experiments, cells were first washed with PBS and starved in HBSS (Gibco Thermo Fisher Scientific, 14025092) for up to 4 h. In some experiments, Bafilomycin A1 (BMLCM110‐0100, Enzo Life Sciences), with DMSO (1:250, Sigma 6768‐5) as a control, were added to the cell media. Bafilomycin A1 was resuspended in DMSO to prepare a 100 μM stock solution, according to the manufacturer's specifications. MRT 68921 (Tocris, 5780) was used for pharmacological inhibition of ULK1. The compound was diluted in autoclaved water to a concentration of 10 mM. For use, the drug was further diluted 1:1000 with HBSS and incubated with the cells for 4 h.

GFP_LC3 HeLa was generated as previously described42 (link). The above-mentioned cells and HEK293 cells were maintained with DMEM (Dulbecco’s modified Eagle’s medium) complemented with 10% fetal bovine serum (FBS, Lonza). To measure mTOR activity upon resveratrol treatment, cells were pretreated with 50 μM resveratrol (Sigma-Aldrich) for 30 min before stimulation with insulin or amino acids. Amino acid or serum fasting was conducted by maintaining cells with HBSS (Hank’s balanced salt solution; Invitrogen) supplemented with 10% dialyzed FBS (Invitrogen) or DMEM only, respectively. Before the resveratrol treatment, 10 μM H-89 (Biomol) was add for 1 h. For autophagy formation analysis using Western blotting, 10 μM of resveratrol was treated into cells in the presence or absence of 10 μM of Bafilomycin A1 (Enzo) for 2 h and cells were subjected to SDS-PAGE and Western blotting.

Primary MEFs at 80% confluency from stably expressing GFP-LC3B WT and Atg4d-deficient mice were incubated in Dulbecco’s modified Eagle’s medium (DMEM, Sigma-Aldrich) complemented with heat-inactivated fetal bovine serum as control condition; in amino acid-free Earle’s balanced salt solution (EBSS, Sigma-Aldrich) to induce autophagy and in EBSS with Bafilomycin A1 (50 nM, Enzo Life Science) to block the autophagy flux. After 4 h of incubation, cells were trypsinized, pelleted by centrifugation, washed with Dulbecco’s Phosphate-Buffered Saline (DPBS), and pelleted again. Cell pellets were resuspended in DPBS supplemented with 5% of heat-inactivated FBS to a density of 105 cells/mL, kept in ice and GFP fluorescence intensity immediately analyzed by Fluorescence-Activated Cell Sorter (BD FACSAria Il, BD Biosciences). Data analysis and graphical representation were done using the free software Flowing Software 2.5.1. The level of the GFP fluorescence intensity was normalized to the level of the control sample, set at 100%. Data represent the mean and SEM of three independent experiments.

Copper (II) sulfate, tetraethylthiuram disulfide (disulfiram), and bathocuproine disulfonic acid disodium salt (BCS) were purchased from Sigma-Aldrich. Elesclomol was purchased from Selleckchem and bafilomycin A1 was purchased from Enzo Life Sciences. Copper (II) sulfate was dissolved in water and stored at room temperature. BCS, Elesclomol, bafilomycin A1, and disulfiram were dissolved in DMSO, aliquoted, and stored at −20°C until use. We used a HP D300 Liquid Dispenser (Tecan) for all experiments involving DMSO-dissolved compounds.

Bone marrow-derived macrophages (BMDM) were generated by plating bone marrow in L929 conditioned medium containing M-CSF in 5 cm diameter non-cell culture treated Petri dishes as described previously [18 (link)]. On day 7, BMDM were harvested in ice cold PBS, 5% FBS, 2.5 mM EDTA by incubating 12 min in the fridge and resuspending by pipetting. The cells were then seeded at 1 x 10⁵ cells/well in 96-well plates and rested overnight prior to infection. L. pneumophila used for infection was grown for 3 days at 37°C on CYE agar plates, then inoculated in ACES yeast extract medium at an OD600 of 0.1 and grown for 21 h at 37°C before use, with 5 mg/ml chloramphenicol added to maintain plasmids. BMDM were infected at MOI 0.1, synchronized by centrifugation, and incubated for 3 days at 37°C, 5% CO₂. Intra- and extracellular CFU were quantified on day 3 by plating on CYE plates after a 10 min incubation in dH₂O to lyse BMDM. Where indicated, 20 nM V-ATPase inhibitor bafilomycin A1 (Enzo Life Sciences, BML-CM110-0100), 25 μM cathepsin B inhibitor CA-074-Me (Enzo Life Sciences, BML-PI126-0001), 25 μM cathepsin D inhibitor pepstatin A (Enzo Life Sciences, ALX-260-085-M005), 2 μg/ml TNFR1-Fc (Adipogen, AG-40B-0074-C050), 25 μg/ml anti-IL1β (R&D, AB-401-NA), 25 μg/ml anti-TNF (Bioxcell, BE0058, clone XT3.11) or 100 ng/ml TNF (Peprotech, 315-01A) were added 15 min prior to infection.