Cells and treatment
A549 and PC9 NSCLC cells from the Cell Bank of Shanghai Institute of Cell Biology, Shanghai, China. BBR and DMSO were from Sigma (MO, USA). Antibodies specific for coxIV, p-JNK, JNK, p-ASK1, ASK1, bax, bcl-2, caspase3, cytochrome c, and β-actin were from Abcam (MA, USA). HRP-labeled anti-mouse and anti-rabbit IgG were from Santa Cruz Biotechnology Inc. (TX, USA). RIPA buffer, Hoechst33342, and DCFH-DA were from Sigma-Aldrich Inc. (MO, USA). JC-1 was from Life Technologies Inc. (CA, USA). A Caspase-3 Colorimetric Assay Kit was from Nanjing Keygen Biotech CO. Ltd. (China). A High Pure Mitochondria Isolation Kit was from Shanghai Genmed Biotech CO. Ltd. (China). All other materials were from BioRad.
A Cell Counting Kit-8 (CCK-8, Dojindo, Kumamoto, Japan) approach was used to monitor cell proliferation. Briefly, 2×103 cells were added to 96-well plates with a range of BBR concentrations (0, 20, 40, 80, and 160 µM). After 24, 48, or 72 h, cells were incubated for 2 h with CCK-8 reagent at 37°C, and absorbance was then measured via spectrophotometer (BioTek, USA).
Cells were treated for 48 h with BBR (0, 40, 80 µmol/L), rinsed with pre-cooled PBS and stained for 20 min with Annexin V-FITC/PI (BD Biosciences, CA, USA) in the dark, followed by analysis via flow cytometry (BD Biosciences).
Intracellular ROS measurement
The membrane-permeable DCFH-DA probe was utilized to quantify ROS levels within cells. Exposure to intracellular esterases results in the inability of hydrolyzed DCFH to exit the cell, while exposure to peroxides results in its oxidation to yield DCF, which is fluorescent. Following treatment with 50µM of BBR, cells were treated for 30 min with DCFH-DA (50 µM). After two washes with PBS, cells were lysed and analyzed via flow cytometry (BD Biosciences).
Mitochondrial membrane potential analysis
Mitochondrial membrane potential (MMP) was assessed with the JC-1 probe. Briefly, cells were initially treated for 48 h with a range of BBR concentrations (0, 40, 80 µM), followed by a 20 min incubation with JC-1 (10 µM) at 37°C in the dark. Cells were then washed prior to analysis via flow cytometry (BD Biosciences, USA), with JC-1 aggregates (red) being detected at 590nm, and JC-1 monomers (green) being detected at 529 nm. The resultant ratio of red to green fluorescence was then reported.
Cellular mitochondria were collected with a High Pure Mitochondria Isolation Kit following a 48 h BBR treatment based on provided instructions. Briefly, 107 cells were rinsed using chilled reagent A, after which they were lysed on ice with the prepared lysis reagent. Samples were then spun at 800g at 4℃ for 10min, after which the supernatants were spun again at 13,000g at 4℃ for 10min to collect mitochondria.
RIPA buffer containing protease inhibitors (NCM Biotech) was used to lyse cells, after which a BCA Protein Assay Kit (Beyotime, Shanghai, China) was used to quantify protein levels. Equal protein amounts from each sample were then separated via 10% SDS-PAGE (Haochen Biotechnology Co., Ltd., Shanghai, China) and transferred to nitrocellulose membranes. Blots were blocked for 1 h with 3% BSA (Google Bio), after which they were probed overnight with anti-β-actin (1:2000), anti-bax, anti-bcl-2, anti-caspase-3, anti-p-JNK, anti-JNK, anti-cytochrome c, and anti-coxIV (1:1000) at 4°C. After three washed in PBST, blots were probed with secondary antibodies for 1h at room temperature and visualized with an Odyssey scanner (LI-COR Biosciences, USA).
Immunohistochemical (IHC) analysis
IHC testing technology was applied to detect the expression level of the candidate target gene. The samples were dewaxed, paraffin-embedded, and incubated in 3% hydrogen peroxide for 30 min to suppress endogenous peroxidase activities. Citrate buffer was used to infiltrate these sections, followed by a 10-min heating process in a microwave oven to retrieve the antigen. Afterwards, these sections underwent an overnight-incubation in primary antibodies (1:2000) at 4°C, rinsed with PBS, treated with peroxidase-labeled goat anti-mouse secondary antibodies at room temperature for 1h, stained with hematoxylin and 3’-diaminobenzidine tetrahydrochloride (DAB), and finally visualized.
Data are given as mean ± SD, and were compared via one-way ANOVAs with posthoc least significant difference tests. The significance threshold for this study was p ≤ 0.05.