br Cell lines and culture conditions br
Cell lines and culture conditions
Human NSCLC cells A549 and non-cancer lung fibroblast cells MRC-5 were originally purchased from Cell Bank of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences (Shanghai, China). It was cultured in RPMI-1640 (Corning) medium containing 10% fetal bovine serum (Gibco) and 100× penicillin-streptomycin solution (Beyotime Biotechnology) at 37 °C in a humidified atmosphere of 5% CO2 incubator. Phytomedicine 52 (2019) 79–88
Reagents and antibodies
Compound 6 was friendly provided by Chen (Guo et al., 2018), and its purity is 97.12% (HPLC analysis, Phenomenex C18 (5 μm particle size silica, 250 mm × 4.6 mm), water-acetonitrile 70:30 as mobile phase with flow rate 1 ml/min, wavelength 254 nm, 25 °C). Other re-agents were purchased as follows: 3-(4, 5-dimetrylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), 3-Methyladenine (3-MA), chlor-oquine (CQ) and ammonium chloride (NH4Cl) (Sigma-Aldrich, St. Louis, MO, USA), LY294002 and necrostatin-1 (Beyotime Bio-technology, Haimen, China), Bafilomycin A1 (Baf A1) and Rapamycin (Sangon Biotech, Shanghai, China), caspase inhibitor z-VAD-fmk, An-nexin V-FITC/PI Apoptosis Detection Kit and BCA Protein Quantitation Kit (KeyGen Biotech, Nanjing, China), Cyto-ID® Autophagy Detection Kit (Enzo Life Sciences, Farmingdale, NY, USA), Lyso Tracker Red dye (Invitrogen, San Diego, CA, USA), Perfluorooctanoic Prostaglandin J2 (PFOA) (Meilun Biotech, Dalian, China). The primary antibodies as well as secondary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). r> Morphological analysis
Cells subjected to treatment with 50 nM compound 6 for indicated times were detected by an inverted microscope (Nikon, Japan).
Cell viability assay by MTT
Cells (5 × 103/well) were seeded in 96-well plates and incubated overnight for attachment. After autophagy inhibitors pretreatment, cells were administered with compound 6 for indicated concentrations and times. Then, the incubation of these cells with MTT (0.5 mg/ml) was implemented in a 37 °C incubator. Thereafter, DMSO (100 μl/well) was added to replace the medium and then to thoroughly dissolve formazan. The absorbance was measured at 570 nm by using a micro-plate reader.
Apoptosis analysis by flow cytometer
Annexin V-FITC/PI Apoptosis Detection Kit was applied to the de-termination of apoptosis. After the planned administration, the ob-tained cells were washed twice with PBS, and then re-suspended in binding buﬀer. Subsequently, cell co-staining with Annexin V-FITC and PI was implemented in the dark at 37 °C for 20 min. Analysis was performed immediately by using a FACS Calibur flow cytometer (Becton-Dickinson, Fullerton, CA, USA).
The obtained cells were washed twice with PBS and lysed thor-oughly in RIPA Cell Lysis Buﬀer (Beyotime Biotechnology, Haimen, China) at 4 °C for more than 30 min, and then centrifuged at 12,000×g for 5 min to get the supernatant. Equal amounts of lysate proteins (20 μg) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene fluoride (PVDF) membranes. Membranes were blocked in TBST sup-plemented with 3%−5% bovine serum albumin (BSA) for 2 h and in-cubated with primary antibodies diluted in blocking buﬀer at 4 °C overnight. Then, they were probed with peroxidase-conjugated sec-ondary antibodies at room temperature for 1.5 h, and the immunoblots were detected using enhanced chemiluminescence system (Pierce, Rockford, IL, USA). The intensities of the resulting protein bands were quantifed by ChemiDoc software (Bio-Rad, USA).