Cell culture, antibodies, regents, and drugs
The tumor cell lines B16F10, A549, EG7, and LL2 were selected for this research. These cells were gifts of Prof. Hongbo Luo, Harvard University, and were maintained in RPMI 1640 (Gibco, Gaithersburg, USA) or DMEM (Life Technologies, CA, USA), each supplemented with 10% fetal bovine serum and penicillin G sodium and streptomycin sulfate (100 units/ml) in a humidified atmosphere of 5% CO2 and 95% air. ABT-737 (Selleckchem, Souffelweyersheim, France) used at 1 μM final concentration. MitoTracker Red CMXRos (M7512) and leptomycin B (LMB) (sc-358688) were purchased from Invitrogen (Eugene, OR, USA) and Santa Cruz Biotechnology (Santa Cruz, CA, USA), respectively. All antibodies including ATF2(C-19, sc-187), p-ATF2(F-1, sc-8398), COX4 (D-20, sc-69359), Bim (N-20, sc-8265), PUMAα (N-19, sc-19187), Bax (P-19, sc-526), cytochrome c (C-20, sc-8385), VDAC1 (B-6, sc-390996), Mcl-1 (S-19, sc-819), β-Actin (C4, sc-47778) were purchased from Santa Cruz Biotechnology. The monoclonal anti-HK1(C35C4, 2024) antibody was obtained from Cell Signaling Technologies (MA, USA). Paclitaxel ((PTX, 33069-62-4) was obtained from Beijing Zhongshuo Pharmaceutical T & D Co., Ltd (Beijing, China). Staurosporine (STS), cisplatin, 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS), carbonyl cyanide m-chlorophenylhydrazone (CCCP), and propidium iodide were purchased from Sigma (St. Louis, MO, USA). The annexin V-FITC kit (K201-100) was purchased from Biovision. PTX was purchased from Calbiochem (La Jolla, CA) and dissolved in 100% dimethyl sulfoxide to produce a stock solution of 1.0 mM.
RNA interference
Cells were transfected at ~70-90% confluence (approximately 1 × 105 cells/ml density). ATF2-specific shRNA clones (ID: TRCN0000013713, Sigma, USA) were obtained from Open Biosystems (catalog no. RHS4533). Lentiviral particles packaged with ATF2 shRNA or scrambled shRNA (control) were generated and spin infected into the target cells in the presence of 10 mg/ml polybrene (Sigma, USA) [15]. The transfection of a synthetic siRNA (25 nM) for VDAC1 was performed with Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. The sense sequence of the double-stranded siVDAC1 siRNA was 5′ AGUGACGGGCAGUCUGGAATT 3′. A scrambled siRNA, which served as a negative control, was purchased from GenePharma (Shanghai, China). HK1 siRNAs (ID: 1599) were obtained from Ambion (USA). The same siRNA reagents were added to the medium at 24 hours post-transfection for 24 hours. The western blot analysis was used to evaluate gene silencing effects. The appropriate controls were included during the entire siRNA knockdown process, confirming the specificity of the siRNA. For the construction of tetracycline-regulated gene expression vectors expressing ATF2T52A mutants (mitochondria localization), DNA was amplified by PCR using pEF-HA-ATF2 (WT, T52A mutants), a gift of Ze’ev A. Ronai (Sanford-Burnham Medical Research Institute, La Jolla, CA 92037, USA), and subcloned into pTHE, resulting in pTHE-WT, ATF2T52A, which were transfected into B16F10 and screened with tetracycline.
Cell viability determination (MTT Assay)
Subconfluent monolayers of the B16 cell line were established in 96-well plates. After overnight incubation, the cells were exposed to different treatments in a medium containing 0.5% fetal bovine serum for 72 hours; 10-μl aliquots of 3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyl-tetrazolium bromide (MTT) solution (10 mg/mL in PBS) were added, followed by 100 μL of 10% sodium dodecylsulfate (SDS) to dissolve the formazan crystals formed. The absorption of the samples was determined using an ELISA reader (Anthos Mikrosysteme GmBH, Germany) at a wavelength of 570 nm. A standard optical density of the untreated control cells was considered at 100% viability. Survival was evaluated by the absorbance of the treated cells normalized to the controls.
Confocal immunofluorescence assays
Cells from different treatment groups were incubated with MitoTracker Red (25 nM) for 15 minutes, fixed, permeabilized, and stained with antibodies for the detection of ATF2 (20 F1). The primary antibodies were revealed using either goat anti-rabbit or anti-mouse IgG conjugated to Alexa 488 (green) (1:500, diluted in blocking solution) from Molecular Probes-Invitrogen. After 1 hours of incubation, the slides were mounted, and the stained cells were analyzed using a confocal microscope (Leica Microsystems Heidelberg GmbH, Heidelberg, Germany).
Apoptosis assay by annexin V/propidium iodide staining
At various time points, control and treated cells were collected following treatment and subjected to apoptosis measurement using the annexin V/propidium iodide (PI) detection kit (R&D Systems) according to the manufacturer’s instructions. A total of 10,000 cells (within whole-cell gates) per replica (3 independent experiments) were subjected to a flow cytometric analysis to evaluate the green fluorescence of annexin V and the red fluorescence of DNA-bound PI. All the data were analyzed with FlowJo software (TreeStar, OR).
Cytochrome c release assay
Isolated tumor cells (5 × 107) were collected and assayed with the Cytochrome c Apoptosis Assay Kit (Cat. #K257-100, Biovision, CA, USA). Briefly, the cells were homogenized with the cytosol extraction buffer provided in the kit and then centrifuged at 700 × g for 10 minutes at 4°C to remove the debris. The supernatant was then centrifuged at 10,000 × g for 30 minutes at 4°C; the pellet contained the mitochondrial fraction, and the supernatant was collected as the cytosolic fraction. These fractions were analyzed for cytochrome c by western blotting using the cytochrome c antibody provided in the kit.
Immunoprecipitation and analysis of protein expression
Cells, transfected as indicated, were lysed in the buffer for 45 min. Lysate aliquots of equal concentration were then incubated overnight with 2 μg of anti-ATF2, −VDC1, −Bim, and -Puma antibodies in an overhead rotator, followed by 20 μl protein G-Sepharose beads (Amersham Pharmacia Biotech, Uppsala, Sweden) for 2 h. The immunoprecipitated proteins were incubated at 70°C for 15 min and analyzed by immunoblotting with conformation-specific primary antibodies against ATF2, VDC1, Bim, Puma, HK1, and VDAC1 (Cell Signaling Technology). β-actin (Chemicon International, Temecula, CA, USA) was performed as loading control.
Cell fractionation
Fractions of cytoplasm nuclear, and mitochondria were separated using a commercial Qproteome mitochondria extraction kit and a Qproteome nucleus extraction kit (Qiagen, Toronto, ON, Canada). Briefly, cells were firstly lysed and centrifuged for 5 min at 1000 × g to remove unbroken cells and nuclei. The supernatant was separated from the pellet and centrifuged at 2,200 × g for 20 min at 4°C to pellet the mitochondria-enriched heavy membrane fraction. The resulting supernatants were combined and further centrifuged at 4°C at 12,000 × g for 30 min at 4°C to obtain the cytoplasmic fraction. An immunoblot analysis was performed as described below.
Western blot analysis
Cells from different treatment groups were lysed using a protein extraction buffer. Total proteins (10 μg) were separated by SDS-PAGE and transferred to nylon membranes (Shanghai Sangon Biotech, Shanghai, China). The blots were hybridized with antibodies indicated above. The secondary antibody, horseradish peroxidase-coupled immunoglobulin (Jingmei Biotech Co., Ltd. Shenzhen, China), was then inculated for 1 h. β-actin (Sigma) was used as loading control. All critical blots and immunoprecipitation experiments were repeated at least three times.
Mitochondrial membrane potential detection
Cells were treated and resuspended in serum-free medium at a concentration of 1 million cells/ml. Each sample was added 5 μl of JC-1 dye (200 μM) for incubation at 37°C, 30 min. The samples were measured by flow cytometry, with 10,000 events collecting. Results were also observed under fluorescence microscopy.
Tumor implantation procedure
C57BL/6 female (8–10 weeks old) mice were purchased from Chongqing Medical University Animal Center (Chongqing, China). All animal experiments were performed with the approval of the Animal Institute Committee. B16F10 cells stably transfected with ATF2 shRNA, ATF2T52A or with empty vector (1.0 × 106/0.1 ml) were injected subcutaneously. The tumor sizes were evaluated using calipers every 2 to 3 days, and the tumor volumes were calculated using the formula: volume = (a2 × b)/2 (a, the short tumor length; b,the long tumor length). In one arm of the experiment, nonnecrotic, single-cell suspensions from tumor tissue were prepared for FACS staining of annexin V/propidium iodide. A portion of the freshly isolated tumor tissue was subjected to a western blotting assay and real-time PCR analysis, as described in the results section.
Statistical analysis
Data are expressed as means ± standard errors of the mean (SEM). Unless indicated otherwise, comparisons were determined using the Student’s t test and one-way ANOVA. P < 0.05 were considered as significance difference.