Bafilomycin a1 bafa1

Human cancer cell line PC-9 was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and genotyped for identity at China Center for Type Culture Collection (CCTCC, Wuhan, China). Gefitinib-resistant PC-9/GR cells were gifted by Prof. Shiyue Li and Dr. Ming Liu from Guangzhou Medical University (Guangzhou, China). H1975 cells were kindly provided by Dr. Xinling Ren from Air Force Military Medical University (Xi’an, China). These cell lines were all cultured with DMEM or RPMI 1640 medium (HyClone, Utah, USA) supplemented with 10% fetal bovine serum (FBS, BI, Israel) at 37C, with 5% CO2. To prepare for further studies, PC-9/GR and H1975 cells were exposed to 2 μM icotinib or DMSO for 4 weeks. Icotinib was provided by Betta Pharmaceuticals Co., Ltd. (Zhengjiang, China). 3-MA, bafilomycin A1 (BafA1), and chloroquine (CQ) were purchased from Sigma Aldrich (Merck KGaA, Darmstadt, Germany). Cryptotanshinon (CTN) was obtained from MCE (USA).

U-251 cells were grown to ~70% confluency for various experiments. Cells were treated with various concentrations of the autophagy inhibitor Bafilomycin A1 (BafA1) (Sigma Aldrich, St. Louis, MO, U.S.A.) or the autophagy inducer Rapamycin (Rapa) (Sigma Aldrich) for various periods of time.

Immortalized mouse embryonic fibroblasts (MEFs) were maintained at 37 °C in a 5% CO₂ atmosphere and cultured in media composed of high glucose DMEM (Thermo Fisher Scientific, Waltham, MA, USA, 10569-010) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific, 16000-044), 100 U mL⁻¹ penicillin and 100 mg mL⁻¹ streptomycin (Gemini, 400-109). For treatments, cell culture media was supplemented with 25 μM carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP) (Sigma-Aldrich, St. Louis, MO, USA, C2920), 50 nM Bafilomycin A1 (BafA1) (Sigma, 19-148), or 100 nM Rapamycin (LC Laboratories, Woburn, MA, USA, R-5000) for the indicated amounts of time. For hypoxia experiments, cells were placed in cell culture media supplemented with 40 mM HEPES (pH 7.4) (Thermo Fisher Scientific, 15630-080) and incubated in hypoxic pouches (GasPak EZ, BD Biosciences, Franklin Lakes, NJ, USA, 260683) equilibrated to 95% N₂, 5% CO₂.

Chemicals used were the following: N-acetylcysteine (NAC; Sigma-Aldrich), Bafilomycin A1 (BafA1; Sigma-Aldrich), Torin1 (Torin1; Calbiochem), Rapamycin (Rapa; Calbiochem), and Cycloheximide (CHX; Sigma-Aldrich).

The drugs and inhibitors used for the current study viz., RSV and TMZ were purchased from Adooq Bioscience (USA). 3MA, Bafilomycin A1 (BaF-A1) and Chloroquine (CQ) were purchased from Sigma (Sigma Aldrich, MO, USA). Z-DEVD FMK and Z-VAD FMK were obtained from Calbiochem (Calbiochem, LaJolla, CA). 3-MA was directly dissolved in DMEM prior to use, CQ was dissolved in sterile water. Baf-A1, Z-DEVD FMK and Z-VAD FMK were dissolved in DMSO to prepare respective stock solutions.

Intracellular killing assays were performed as described previously (Munzenmayer et al., 2016 (link)). For S. aureus infection, early stationary phase bacteria (OD_600nm = 1.0–1.5) were harvested and washed once in cold phosphate-buffered saline (PBS). RAW264.7 cells were then infected with S. aureus at a multiplicity of infection (MOI) of 10. Following incubation for 1 h, infected cells were washed three times with PBS before the addition of 10% (v/v) FCS-DMEM supplemented with 10 μg/ml lysostaphin (Sigma-Aldrich) and 100 μg/ml gentamicin (Sigma-Aldrich) to each well. Plates were then incubated for 1 h to kill extracellular bacteria. Following incubation, the cells were washed with PBS and further incubated in fresh 10% (v/v) FCS-DMEM. At 0, 3, 6, 12, and 24 h post-infection (hpi), infected cells were washed three times with PBS to remove extracellular bacteria and dead cells and lysed by the addition of 0.5% (v/v) Triton X-100 (Sigma-Aldrich). The number of intracellular bacteria (expressed as colony-forming units, CFU) was determined by serial dilution and plating on TSB agar. In addition, replicate plates were incubated with 1.25 mM 3-methyladenine (3-MA, Sigma) or 100 nM bafilomycin A1 (Baf A1, Sigma) for 2 h prior to infection to block autophagy in the RAW264.7 cells.