Cells and reagents
Human lung epithelial cancer H460 cells and immortalized human normal lung epithelial BEAS-2B cells were obtained from the American Type Culture Collection (Manassas, VA). The cells were cultured in RPMI 1640 and DMEM medium containing 10% fetal bovine serum (FBS), 2 mM l-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin in a 5% CO2 environment at 37°C, respectively. H2O2, NAC, GSH, catalase, and antibody for ubiquitin were obtained from Sigma-Aldrich (St. Louis, MO). Hoechst 33342, annexin V-FITC, H2DCF-DA, and Alexa Fluor secondary antibodies were obtained from Molecular Probes (Grand Island, NY). Lactacystin, concanamycin A, PD98059, and U0216 (MAP kinase ERK1/2 inhibitors), SP600165 (c-Jun N-terminal kinase inhibitor), and SB203580 (p38 kinase inhibitor) were from Calbiochem (La Jolla, CA). Antibodies for Bcl-2, phospho-ERK1/2 (p-p44/p42), ERK1/2 (p44/p42), horseradish peroxidase (HRP)–conjugated secondary antibodies, and HRP-conjugated anti-biotin antibody were obtained from Cell Signaling Technology (Beverly, MA). Antibody for cysteine sulfenic acid was obtained from Millipore (Billerica, MA). DCP-Bio1 was obtained from KeraFAST (Boston, MA).
Plasmids and transfection
Bcl-2 and mutant plasmids were generously provided by C. Stehlik (Northwestern University, Chicago, IL). The authenticity of the plasmid constructs was verified by DNA sequencing. Cells were transfected with hemagglutinin-tagged wild-type Bcl-2, Bcl-2 mutant, or pcDNA3 control plasmid by nucleofection using Nucleofector (Amexa Biosystems, Cologne, Germany), according to the manufacturer's instructions. Briefly, cells were suspended in 100 μl of nucleofection solution with 2 μg of plasmid and nucleofected using the device program T020. The cells were then resuspended in 500 μl of complete medium and seeded in 6-mm cell culture dishes. Cells were allowed to recover for 48 h before each experiment. The efficiency of transfection was determined by using a green fluorescent protein reporter plasmid and was found to be ~80%.
Apoptosis was determined by Hoechst 33342 assay and by flow cytometric analysis of annexin V and propidium iodide (PI). In the Hoechst assay, cells were incubated with 10 μg/ml Hoechst 33342 for 30 min and analyzed for apoptosis by scoring the percentage of cells having condensed chromatin and/or fragmented nuclei by fluorescence microscopy (Leica Microsystems, Bannockburn, IL). Approximately 1000 nuclei from 10 random fields were analyzed for each sample. The apoptotic index was calculated as the percentage of cells with apoptotic nuclei over total number of cells. For flow cytometric analysis, cells were harvested, washed, stained with annexin V–fluorescein isothiocyanate (FITC) in binding buffer for 30 min at room temperature, and costained with PI (10 μg/ml). Samples were immediately analyzed by FACScan flow cytometer (Becton Dickinson, Rutherford, NJ) using a 488-nm excitation beam and a 530- and 630-nm band-pass filter with CellQuest software (Becton Dickinson).
ROS generation was determined fluorometrically using H2DCF-DA as a fluorescent probe. Briefly, cells were incubated with the probe (10 μM) for 30 min at 37°C, after which they were washed and resuspended in phosphate-buffered saline (PBS), followed by analysis of cellular fluorescence intensity by a fluorescence plate reader at the excitation and emission wavelengths of 485 and 538 nm, respectively.
Western blot analysis
After specific treatments, cells were incubated with lysis buffer containing 20 mM Tris-HCl (pH 7.5), 1% Triton X-100, 150 mM sodium chloride, 10% glycerol, 1 mM sodium orthovanadate, 50 mM sodium fluoride, 100 mM phenylmethylsulfonyl fluoride, and a commercial protease inhibitor mixture (Roche Molecular Biochemicals, Indianapolis, IN) at 4°C for 20 min. Cell lysates were collected and determined for protein content using the Bradford method (Bio-Rad Laboratories, Hercules, CA). Proteins (40 μg) were resolved under denaturing conditions by 10% SDS–PAGE and transferred onto nitrocellulose membranes. The transferred membranes were blocked for 1 h in 5% nonfat dry milk in TBST (25 mM Tris-HCl, 125 mM NaCl, 0.05% Tween-20) and incubated with appropriate primary antibodies at 4°C overnight. The membranes were washed twice with TBST for 10 min and incubated with HRP-conjugated secondary antibodies for 1 h at room temperature. The immune complexes were then detected by chemiluminescence detection system (Amersham Biosciences, Piscataway, NJ) and quantified using Analyst/PC densitometry software (Bio-Rad Laboratories).
After specific treatments, cells were washed with PBS and lysed in lysis buffer at 4°C for 20 min. The lysates were collected and determined for protein content using the Bradford method (Bio-Rad Laboratories). Cell lysates (60 μg protein) were incubated with anti–Bcl-2 or anti-ERK1/2 (p44/p42) antibody at 4°C for 14 h, followed by incubation with protein G–conjugated agarose for 4 h at 4°C. The immune complexes were washed six times with cold lysis buffer and resuspended in 2× Laemmli sample buffer. The immune complexes were separated by 10% SDS–PAGE and analyzed for protein expression by Western blotting.
Cysteine sulfenic acid detection
Cysteine sulfenic acid was determined by Cys-SOH antibody–based and DCP-Bio1 reagent–based assays. In the antibody-based assay, cells were washed with PBS and lysed in lysis buffer containing 0.1 mM dimedone at 4°C for 20 min. Cell lysates (200 μg of protein) were immunoprecipitated using anti–Bcl-2 antibody and separated by 10% SDS–PAGE as described. Cysteine oxidation was detected by Western blotting using 1:1000 anti–cysteine sulfenic acid antibody (Millipore, MA) and anti-rabbit HRP-conjugated secondary antibody. For the reagent-based assay, cells were labeled with 1 mM cysteine oxidation probe DCP-Bio1 (KeraFAST), which is a dimedone analogue containing biotin tag. The DCP-Bio1–labeled proteins were similarly immunoprecipitated using anti–Bcl-2 antibody and separated by 10% SDS–PAGE as described. Subsequently, the samples were detected for cysteine oxidation by Western blotting using 1:1000 anti-biotin (conjugated HRP) antibody. Quantification of the immune signals was performed using Analyst/PC densitometry software.
Cells were seeded on rat type I collagen–coated coverslips (5 μg/cm2), fixed with 3.7% paraformaldehyde for 15 min, incubated in 50 mM glycine for 5 min, permeabilized, and blocked with 0.5% saponin, 1.5% bovine serum albumin, and 1.5% normal goat serum for 30 min. ERK was immunostained with anti-ERK1/2 antibody, and Bcl-2 was immunostained with anti–hemagglutinin tag antibody. Secondary Alexa Fluor 488–, 546–, and 647–conjugated antibodies and phalloidin (Invitrogen, Carlsbad, CA) were used. Cells were washed with PBS containing 0.5% saponin and were mounted on a coverslip using Fluoromount-G (Southern Biotechnology Associates, Birmingham, AL). Cells were visualized with a Zeiss LSM 510 confocal on an AxioImager Z1 microscope using a 63× objective lens (Carl Zeiss, Jena, Germany).
The data represent means ± SD from three or more independent experiments. Statistical analysis was performed by Student's t test at a significance level of p < 0.05.