Reagents and cell lines
All chemical reagents were purchased from Sigma-Aldrich Corporation (St. Louis, MO.) unless otherwise specified. The experimental, small-molecule, Bcl-2 BH-3 domain inhibitor A-779024 was provided by Abbott Laboratories (Abbott Park, IL). The pan-Caspase inhibitor ZVAD-FMK was purchased from MBL International (Woburn, MA). Monoclonal antibodies to Akt, phospho-Akt, Beclin 1, Mcl-1, and LC-3 were purchased from Cell Signaling (Beverly, MA). Bax and Bcl-2 antibodies were purchased from BD Pharmingen (San Diego, CA). Caspase 3 antibody was purchased from Biosource (Camarillo, CA); polyclonal antibodies to Bcl-xL and actin was purchased from Santa Cruz Biotechnology (Santa Cruz, CA).The MiaPaca-2 cell line was obtained from the American Type Culture Collection (Rockville, MD) and cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, sodium pyruvate, nonessential amino acids, L-glutamine, vitamins, penicillin, and streptomycin. Cells were maintained at 37°C in a humidified incubator containing 5% CO2.
Cell Cycle Analysis with Flow Cytometry
1 × 106 cells per treatment were plated in 100mm plates and allowed to recover overnight. Following treatments, cells were harvested with trypsin, washed in PBS, and resuspended in 150uL of FACSMAX solution (Genlantis, San Diego, CA) and incubated 10 minutes on ice to maximally disperse the cells. They were then fixed by adding ice cold 70% ethanol dropwise and kept at +4 degrees Celsius 1–7 days. Prior to analysis, cells were washed with PBS, then incubated with Ribonuclease A (200 mcg/ml) for 30 minutes at 37 degrees Celsius, and then stained with propridium iodide (10 mcg/ml). The cells were then analyzed using a Becton Dickenson FACscan (BD Biosciences, San Jose, CA) to determine the DNA fractions and the Sub-G0 (Apoptotic) cell population was quantified using Flowjo software (Treestar, inc., Ashland, OR).
Live Cell Fluorescence Microscopy for Autophagy
A MiaPaca-2 clonal population overexpressing eGFP-LC-3 was generated as previously described. These cells were seeded in Ibidi 8-chamber ultra-thin culture slides (Integrated BioDiagnostics; Munich, Germany) and allowed to recover for 48 hours under standard culture conditions. Culture medium was then changed to include either A-779024 2 µM or Rapamycin 2µM and the cells were incubated for 1 or 6 hours before they were imaged using a IX-71 Delta Vision (Applied Precision; Issaquah, Washington) inverted fluorescent microscope with a 60× 1.40 NA oil objective and FITC filter (excitation, 480 nm; emission, 535 nm). During imaging the cells were kept at 37 degrees Celsius with an ASI 400 air stream incubator (Nevtek; Williamsville, VA).
MiaPaca-2 cells were cultured and treated with specified treatments in 4-well LAB-TEK® chamber slides coated with poly-L lysine (Nalge Nunc International, Naperville, IL), then fixed first in 4% paraformaldehyde (1 min at room temperature) and then 100% ice-cold Methanol (10 minutes). Cells were washed in Phosphate buffered saline (PBS) and the cell membranes solubilized by incubation in PBS with 0.5% Triton X-100 for 20 min at room temperature. All incubations were done in a humidity chamber. Blocking of nonspecific binding was achieved by incubation in 0.1% Triton X-100 with 2% bovine serum albumin for 30 minutes at room temperature. Immunostaining consisted of incubation with monoclonal antibodies to BCL-2 (Pharmingen, 1:1000 dilution) and Beclin 1 (Cell Signaling, 1:500 dilution) overnight at 4 C. After 3 washes with 0.1% Triton X-100 in PBS slides were incubated with species-specific secondary antibodies (Alexa FluorR 488 anti-rabbit for Beclin 1, Alexa FluorR 647 anti-hamster for BCL-2; [Invitrogen, Carlsbad, CA]) for 1 hour at room temperature. The plastic chambers were then removed and coverslips mounted using Slowfade Gold® mounting medium (Invitrogen, Carlsbad, CA) containing 4,6-diamidino-2-phenylindole dihydrochloride (DAPI) for nuclear staining. Slides were visualized with a IX-71 Delta Vision inverted fluorescent microscope equipped with a 60× 1.40 NA oil objective (Olympus, Melville, NY) using DAPI, FITC, and CY-5 filters. The signal intensities through each filter were matched at the time of imaging, and the images through each filter were individually optimized for brightness and background prior to generating the final composite images.
Following treatments, cells were harvested with trypsin, pelleted by centrifugation, washed with PBS once, and then resuspended in lysis buffer (Cell Signaling #9803). Total lysate protein was quantified for equal gel loading using the BioRad protein assay (BioRad Labs, Hercules, CA) Lysates were mixed with a reducing loading buffer and heated to 100 degrees Celsius for 10 minutes, resolved by SDS-PAGE, and separated proteins were then electrophoretically transferred to 0.2-mm nitrocellulose membranes (Schleicher & Schuell; Keene, NH). The blots were probed overnight with primary antibodies, and developed using species-specific secondary antibodies conjugated to horse-radish peroxidase. Immunoreactivity was detected by the enhanced chemiluminescence technique (Amersham; Piscataway, NJ).
MiaPaca-2 cells were cultured under standard conditions to near-confluence, then harvested with Trypsin, pelleted by centrifugation, and washed with phosphate buffered saline. The cell pellet was lysed in an immunoprecipitation buffer (50 mM Tris [pH 7.5], 150 mM NaCl, 1% NP40) on ice for 5 min and clarified by centrifugation (10,000 g, 10 min). One mg of total protein used to immunoprecipitate Bcl-2, employing an immobilized monoclonal antibody to Bcl-2 (Santa Cruz Biotechnology, Santa Cruz, CA), which was incubated with the lysate for 45 min at 25°C and isolated using Protein A-agarose. Proteins were then immunoblotted as described above.