Cell lines
Lung adenocarcinoma A549 cells were purchased from Cell Bank of Type Culture Collection of Chinese Academy of Sciences (Shanghai, China). The cells were cultured in RPMI 1640 media supplemented with 10% fetal bovine serum and grown in a humidified incubator at 37 ℃ in a humidified 5% CO2 atmosphere.
MTS assays
A549 cells were seeded at 2000 cells/well in 96-well plates and incubated for 12 h at 37 ℃. And then the medium was replaced with a fresh medium containing various concentrations of cisplatin (0, 0.01, 0.1, 1, 10, 100 µM) and different concentration of resistin (0, 12.5, 25, 37.5, 50 ng/ml). After 48 h of incubation, 20 µl of CellTiter 96 Aqueous One Solution Reagent (Promega, Madison, Wl) was added to each well and incubated for 1.5 h. After that, the absorbance values were determined at 490 nm in Eon Microplate Spectrophotometer (BioTek. Instruments. Inc, Winooski, VT). Each experiment was conducted in triplicate.
Flow-cytometric analyses of apoptosis and cell cycle
Flow cytometry was adopted to analyze the apoptosis rate and cell cycle distribution of cells. A549 cells were seeded in a 6-well plate and treated with cisplatin or cisplatin + resistin. After 48 hours, the cells were collected by trypsin. For apoptosis analysis, cells were stained with FITC-Annexin V and Propidium Iodide (Beyotime, Jiangsu, China), and analyzed using FC500 Flow Cytometer (Beckman Coulter, Fullerton, CA) with CXP software (Beckman Coulter, Fullerton, CA). For cell cycle analysis, cells were stained with Propidium Iodide (Sigma, St. Louis, Missouri, USA) according to the manufacturer’s protocol.
Western Blotting
A549 cells were seeded in a 6-well plate and treated with vehicle, cisplatin, resistin, or cisplatin + resistin for 48 h. Cells were lysed using mammalian protein extraction reagent RIPA (Beyotime, Jiangsu, China) supplemented with protease inhibitor cocktail (Sigma-Aldrich, St. Louis, Missouri, USA). The concentration of total protein was measured using a BCA protein assay kit (Beyotime, Jiangsu, China). Samples (30-50μg protein/lane) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel (Beyotime, Jiangsu, China) and then transferred onto the polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). The membranes were incubated with primary antibodies (primary antibody of cleaved caspase 3 (1:1000, Wanleibio, Shenyang, China), caspase 3 (1:1000, Wanleibio, Shenyang, China), Bcl2 (1:1000, Wanleibio, Shenyang, China), β-actin (1:10000, Sigma, St. Louis, USA)) at 4 ℃ overnight and then incubated with horseradish peroxidase-conjugated secondary antibodies for 1 hour. Protein bands were visualized with ECL substrates (GE Healthcare Bio-Sciences, NJ, USA) using the Western-Light chemiluminescent detection system (Bio-Rad, Hercules, CA, USA) according to the protocols.
Assays of the release of cytochrome C from mitochondria
A549 cells were seeded in a 6-well plate and treated with cisplatin or cisplatin + resistin. After 48 hours, the cells were harvested by trypsin. Fractionation of mitochondrial and cytosolic fractions was conducted using the Cell Mitochondria Isolation Kit (Beyotime, Jiangsu, China) according to the protocol. Then, a western blot assay was adopted to assess the protein levels of cytochrome C (primary antibody: 1:1000, Wanleibio, Shenyang, China) in the mitochondria and cytosol as described above. The distribution of cytochrome C was also evaluated by immunofluorescence assay. Briefly, cells were fixed with 4% paraformaldehyde and incubated with primary antibody against cytochrome C (1:1000, Wanleibio, Shenyang, China) overnight. After washed, cells were immunoblotted with Alexa Fluor-488 conjugated anti-rabbit secondary antibody (Solarbio, Beijing, China) and counterstained with 4'-6-Diamidino-2-phenylindole (Beyotime, Jiangsu, China). After that, the cells were pictured with a Leica fluorescence microscope (Leica, Wetzlar, Germany).
Measurement of ROS production
Flow cytometry and immunofluorescence were used to assess intracellular ROS level with the fluorescent probe of 2’,7’-dichlorofluorescein diacetate (DCFH-DA, Beyotime, Jiangsu, China). Cells were incubated with 10 µM DCFH-DA for 20 min at 37 ℃ and then washed with PBS triple. For flow cytometry analysis, cells were collected by trypsinization and resuspended in 0.5 ml of PBS. The detection wavelength for DCFH-DA was 525 nm. For immunofluorescence, cells were washed and photographed by a Leica fluorescence microscope (Leica, Wetzlar, Germany).
Measurement of mitochondrial membrane potential (MMP)
Flow cytometry and immunofluorescence were used to assess MMP with the fluorescent probe of 5,5′,6,6′-Tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine iodide (JC-1, Beyotime, Jiangsu, China). Cells were incubated with JC-1 for 20 min at 37 ℃, then washed with PBS triple. For flow cytometry analysis, cells were collected by trypsinization and resuspended in 0.5 ml of PBS. The detection wavelength for monomeric JC-1 was 525 nm. For immunofluorescence, cells were washed and photographed by a Leica fluorescence microscope (Leica, Wetzlar, Germany).
Measurement of mitochondrial number
Flow cytometry and immunofluorescence were used to assess the number of mitochondria with the fluorescent probe of Mito-Tracker Green (Beyotime, Jiangsu, China). Cells were incubated with Mito-Tracker Green for 30 min at 37 ℃, then washed with PBS triple. For flow cytometry analysis, the cells were collected by trypsinization and resuspended in 0.5 ml of PBS. The detection wavelength for Mito-Tracker Green was 525 nm. For immunofluorescence, cells were washed and photographed by a Leica fluorescence microscope (Leica, Wetzlar, Germany).
Statistical analyses
All data were represented as mean ± standard deviation (SD) of at least three independent experiments. Pearson’s Х2 test or Fisher’s exact test were applied to analyze differences for qualitative variables. Student’s t-test or one-way ANOVA was used for continuous variables. All tests were 2-sided, and P < 0.05 was considered significant. PASW Statistics v18.0 (IBM Co., Armonk, NY, USA) was used for data analysis.