Wild type (WT) and Bax/Bak double knockout (DKO) mouse embryonic fibroblast (MEF) cells are courtesy of Dr. SJ Korsmeyer's lab (Dana-Farber Cancer Institute, Boston, MA) and maintained in Dulbecco's modified Eagle's medium supplemented with 10% heat inactivated fetal bovine serum, 0.1 mmol/L nonessential amino acids, 0.1 μmol/L 2-mercaptoethanol, and antibiotics at 37°C in an atmosphere of 95% air and 5% CO2.
Oxidation-dependent fluorogenic dye, dihydroethidium (DHE) was used to evaluate intracellular production of superoxide radicals. Briefly, cells were incubated with 5 μM DHE for 30 min at the indicated time point. Cells were collected by trypsinization and resuspended in PBS. The fluorescence of ethidium was measured using a FACScan flow cytometer (Becton-Dickinson, Rutherford, NJ) supplied with the CellQuest software. Mean fluorescence intensity from 10,000 cells was acquired using a 585/42 nm bandpass filter.
HPTLC separation of phospholipids and CL oxidation assay
Lipids were extracted from cells and resolved by 2D HPTLC as previously described [13
]. Spots of phospholipids were scraped from the HPTLC plates and phospholipids were extracted from silica. Lipid phosphorus was determined by a micro-method [13
]. CL hydroperoxides were determined by fluorescence HPLC of products formed in Microperoxidase (MP)-11-catalyzed reaction with a fluorogenic substrate, Amplex Red. Oxidized phospholipids were hydrolyzed by pancreatic phospholipase A2 (2 U/ml) in 25 mM phosphate buffer containing 1 mM CaCl2
, 0.5 mM EDTA and 0.5 mM SDS (pH 8.0 at RT for 30 min). After that Amplex Red and MP-11 were added and samples were incubated for 40 min at 4°C. Shimadzu LC-100AT vp HPLC system (Shimadzu, Columbia, MD) equipped with fluorescence detector (RF-10Axl, Excitation/Emission=560/590 nm) and autosampler (SIL-10AD vp) were used for the analysis of products separated by HPLC (Eclipse XDB-C18 column, 5 μm, 150×4.6 mm, Agilent Technology, Santa Clara , CA). Mobile phase was composed of NaH2
(25 mM, pH 7.0)/methanol (60:40 v/v).
Harvested cells were resuspended in lysis buffer containing 250 mM sucrose, 20 mM HEPES-KOH, pH 7.5, 10 mM KCl, 1.5 mM MgCl2, 1mM EDTA, 1 mM EGTA, 1 mM DTT, 1 mM PMSF, 1 μg/mL aprotinin, 1 μg/mL leupeptin, and 0.05% digitonin for 4 min on ice, and then centrifuged at 10,000×g for 5 min. The resulting supernatants and pellets were subjected to 12% SDS-PAGE and then transferred to a nitrocellulose membrane, which was probed with antibodies against Bax (clone 6A7, Pharmingen, San Diego, CA) or cyt c (clone 7H8.2C12, Pharmingen) followed by horseradish peroxidase-coupled detection. Duplicated membranes were probed with actin (anti-actin antibody, Calbiochem, San Diego, CA) or COX-IV (cyt c oxidase subunit IV, anti COX-IV antibody, Abcam, Cambridge, MA) as loading controls, respectively. Semi-quantitation of the bands was carried out by densitometry using Labworks Image Acquisition and Analysis Software (UVP, Upland, CA). The levels of Bax were expressed as the mean densitometry ratio of Bax over actin or COX-IV and then normalized to control.
Phosphatidylserine (PS) externalization
Externalization of PS was analyzed by flow cytometry using annexin-V kit (Biovision, Mountain View, CA). Briefly, harvested cells were stained with annexin-V-FITC and PI for 5 min in dark prior to flow cytometry analysis. Ten thousand events were collected on a FACScan flow cytometer supplied with CellQuest software.
Mitochondrial fractions were prepared by differential centrifugation. Briefly, MEF DKO cells were suspended in mitochondria isolation buffer (MIB, 210 mM mannitol, 70 mM sucrose, 10 mM HEPES, pH 7.4, 1 mM EDTA) and lysed by Dounce homogenization. Unbroken cells, nuclei and debris were removed by 10 min of centrifugation at 700×g at 4°C. A mitochondria rich fraction was obtained by 10 min centrifugation at 5,000×g, and washed twice with MIB.
Recombinant Bax induced release of pro-apoptotic factors from isolated mitochondria
Isolated mitochondria (1 mg/mL) were resuspended in the incubation buffer A (210 mM sucrose, 70 mM KCl, 10 mM HEPES, pH7.4, 3 mM sodium phosphate, 0.5 mM EGTA) in the presence or absence of 5 mM succinate, and then incubated with mouse recombinant Bax (200, 400 nM, ProteinX Lab, San Diego, CA) for 20 min at room temperature. Samples were then centrifuged at 10,000×g for 5 min. The resulting supernatants and pellets were analyzed for cyt c, Smac/Diablo (clone 7, Pharmingen) or COX-IV (loading control to confirm equal amount of mitochondria was used) content by Western Blot. The levels of released proteins were expressed as the mean densitometry ratio of cyt c or Smac/Diablo over COX-IV and then normalized to control sample without Bax incubation.
Mitochondrial peroxidase activity
Peroxidase activity in mitochondria was evaluated using 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA). Mitochondria (0.5 mg/mL) were resuspended in incubation buffer A with 100 μM DTPA and 10 μM DCFH-DA. Recombinant and Bax (200 nM) was then added to mitochondria for 15-min incubation. The reaction was initiated by adding 20 μM H2O2 to the samples. Fluorescence spectra were recorded with a Shimadzu RF-5301PC fluorescence spectrophotometer (Shimadzu, Kyoto, Japan) after 30 min incubation (Excitation=488 nm).
All data are expressed as means ± S.D. of at least three independent experiments. Statistical comparisons between groups were performed by Student's t-test.