Chemicals and reagents
Tiotropium and olodaterol were purchased from Tocris (Ellisville, MO, USA). Acridine orange (N,N,N',N'-tetramethylacridine-3,6-diamine), 5,5’,6,6’-tetrachloro-1,10,3,3’-tetraethylbenzimidazolylcarbocyanine iodide (JC-1; Sigma-Aldrich, St. Louis, MO, USA), and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from (St. Louis, MO, USA). All the chemicals were dissolved in DMSO and stored at −20 °C. MTT, JC-1, and acridine orange were dissolved in water and stored at 4 t .
Anti-LC-3 and Anti-Beclin 1 were purchased from Novus Biological (Littleton, CO, USA). Anti-phospho-ERK and anti-phospho-JNK were purchased from Santa Cruz (Santa Cruz, CA, USA). GAPDH was purchased from Cell signaling (Danvers, MA, USA). Goat anti-rabbit IgG and goat anti-mouse IgG were purchased from Jackson Laboratory (Bar Harbor, ME, USA).
BEAS-2B human lung epithelial cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum (Gibco, Gland Island, NY, USA) and 1% antibiotic antimycotic. The cells were maintained in an incubator at 37 °C with a humidified atmosphere with 5% CO2. The cells were grown to 90% confluence and passaged by trypsin/EDTA.
Cigarette smoke extraction
CSE was prepared as previously described with minor modifications [13, 14]. In brief, commercial cigarettes (LONG LIFE, Taiwan), containing 1.2 mg of nicotine and 12 mg of tar, were used to obtain CSE. The smoke of five cigarettes was bubbled through 10 mL of PBS. The solution was considered to have 100% strength CSE. The peristaltic pump was equilibrated at a rate of one cigarette per 5 min. The CSE suspension was then filtered through a 0.22-µm pore filter to remove bacteria and particles.
Cell viability assay
BEAS-2B cells were seeded in 24-well plates at a density of 2 × 104 cells/mL and were pretreated with or without various concentrations of tiotropium/olodaterol for 4 h. The cells were then cultured with 0%–10% CSE for 24 h. After 24-h incubation, 200 μL of 1 mg/mL MTT in RPMI 1640 was added to each well at 4 h before the end of each incubation. The MTT solution was then removed. The adherent cells were lysed with 600 μL of DMSO, and the optical density was obtained at 570 nm using a microplate reader (PerkinElmer).
Detection of the mitochondrial membrane potential
BEAS-2B cells were seeded in 6-well plates at a density of 2 × 105 cells/mL and pretreated with or without various concentrations of tiotropium/olodaterol for 4 h. The cells were then cultured with 5% CSE for 24 h. After incubation, the cells were removed from the plate with trypsin-EDTA (GIBCO-BRL), and 10 µg/mL JC-1 (Sigma-Aldrich, St. Louis, MO, USA) and serum-free medium were mixed at a ratio of 1:500, and 1 mL of the mixture was added to each sample, followed by incubation at 37 °C for 15 min. The samples were detected and quantified using an Aria III flow cytometer (BD Biosciences).
Annexin V assay
BEAS-2B cells were cultured at a density of 2 × 105 cells/mL in a 6-well plate (Corning Glass Works, Corning, NY, USA). The next day, the cells were then pretreated with 10 μM tiotropium/olodaterol combined 12.5 or 25 μM tiotropium for 4 h followed by 5% CSE exposure for 24 h. After the incubation period, the cells were collected by trypsin-EDTA treatment and centrifugation. Apoptotic cell quantification was performed using an annexin V-FITC apoptosis detection kit (BD Biosciences, Franklin Lakes, NJ, USA). The cells were stained following the annexin V-FITC/PI staining protocol for 15 min at RT. The fluorescence of annexin V/PI was then detected with the Aria III flow cytometer (BD Biosciences).
Acridine orange assay
Autophagy is characterized by the formation of acidic vesicular organelles (AVOs). We used acridine orange to characterize the acidic cellular compartment that emits bright red fluorescence in acidic vesicles and green fluorescence in the cytoplasm and nucleus. BEAS-2B cells were seeded in 6-well plates at a density of 2 × 105 cells/mL and pretreated with and without various concentrations of tiotropium/olodaterol for 4 h. The cells were then cultured with 5% CSE for 6 and 24 h. After incubation, the cells were removed from the plate with trypsin-EDTA (GIBCO-BRL). Acridine orange was added at a final concentration of 1 mg/mL, and the plates were then incubated at 37 °C for 15 min. The AVOs in BEAS-2B cells were detected and quantified using the Aria III flow cytometer (BD Biosciences). The AVOs displayed bright red fluorescence (650 nm, FL-3 channel), whereas the cytoplasm and nucleolus displayed green fluorescence (500–550 nm, FL-1 channel) . The intensity of red fluorescence was proportional to the number of AVOs in autophagic cells.
The cells were lysed in RIPA buffer with the 10% proteasome inhibitor. The cell extracts were cleared at 12000 rpm in a microcentrifuge at 4 °C for 20 min. Proteins were separated by 10% and 15% SDS-PAGE and transferred onto a PVDF membrane. The membrane was blocked in 5% nonfat dry milk in Tris-buffered saline with Tween 20 (TBST) buffer. Immunostaining was then performed using anti-LC-3, anti-Beclin 1, anti-phospho-ERK, or anti-phospho-JNK, followed by incubation with HRP-conjugated anti-rabbit or anti-mouse IgG secondary antibodies. ECL reagent (GE Healthcare Life Sciences, Chalfont, UK) was used for protein detection.
All values are expressed as mean ± SD. For comparison between two groups, we used unpaired two-tailed t tests (Student’s t tests). P values of < 0.05 were considered statistically significant. Data were analyzed using GraphPad Prism Version 6.0 (San Diego, CA, USA).