Dulbecco’s modified Eagle’s medium (DMEM) was purchased from Hyclone (Logan, UT). OptiMEM reduced serum medium was purchased from Gibco (Grand Island, NT). Fetal bovine serum (FBS) was purchased from Atlas Biologicals (Fort Collins, CO). Protease inhibitor cocktail tablets (Complete Mini, EDTA-free) were from Roche (Mannheim, Germany). Caspase inhibitor, Q-VD-OPh, was purchased from R&D Systems (Minneapolis, MN). Lipofectamine2000 reagent, trypsin, penicillin-streptomycin solution, and glutamine were purchased from Invitrogen (Carlsbad, CA). N-Methyl-N’-nitro-N-nitrosoguanidine (MNNG) was purchased from AccuStandard (New Haven, CT). Epirubicin, cisplatin and cyclophosphamide were all purchased from Calbiochem (La Jolla, CA). ApoScreen annexin V-fluorescein isothiocyanate (FITC) conjugate and propidium iodide (PI) were purchased from Southern Biotech (Birmingham, AL). Primary antibodies utilized were polyclonal anti-PAR clone 96–10 (Trevigen, Gaithersburg, MD), polyclonal anti-β-actin (Thermo Fisher Scientific), polyclonal anti-caspase-3 (Millipore, Billerica, MA), monoclonal anti-PARG (Millipore), polyclonal anti-AIF (Rockland Immunochemicals, Gilbertsville, PA), polyclonal anti-manganese superoxide dismutase (MnSOD) (Millipore), monoclonal anti-Lamin B2 clone LN43 (Thermo Fisher), and monoclonal anti-caspase-8 (Cell Signaling, Danvers, MA). Secondary antibodies horseradish peroxidase (HRP)-conjugated goat anti-rabbit and anti-mouse were purchased from Sigma (St. Louis, MO).
Human breast adenocarcinoma cells (MDA-MB-231 and MCF-7) were cultured in DMEM supplemented with 10% FBS, 2 mM L-glutamine and 100U/ml penicillin/streptomycin at 37°C in 5% CO2. Wild type and PARG-null primary embryonic trophoblast stem (TS) cells were derived from E3.5 mouse blastocysts and cultured as previously described . For siRNA transfections, each cell line was cultured at least one day in antibiotic-free medium before transfection.
The silencing of AIF by RNAi was performed as previously reported using sense (5’-CUUGUUCCAGCGAUGGCAUtt-3’) and antisense (5’-AUGCCAUCGCUGGAACAAGtt-3’) RNA oligos that target nucleotides 151–171 in human AIF . The silencing of PARG by RNAi was performed as previously reported using RNA oligos 5’-AAATGGGACTTTACAGCTTTG-3’) that target nucleotides 1960–1980 in human PARG . Control oligos 5’-AGACAGAAGACAGAUAGGCtt-3’ (sense) and 5’-GCCUAUCUGUCUUCUGUCUtt-3’ (antisense) were used for all negative controls . All RNA oligos were purchased as duplexed RNA from Sigma-Aldrich (St. Louis, MO). Each siRNA duplex was resuspended in RNAase-free H2O at a final concentration of 20 μM and stored at −20°C.
For RNAi transfections, cells were plated in 0.5 ml medium per well without antibiotics in 24-well plates one day before transfection. At the time of transfection, cells were approximately 50% confluent. For each transfection, duplex siRNA was added to 50 μl OptiMEM medium and 1 μl lipofectamine2000 was added to 50 μl OptiMEM. After 5 min, the two solutions were gently mixed and then incubated at room temperature for 20 min. Final concentrations of siRNA were 20 nM for AIF and 40 nM for PARG. The mixtures were added to each well and cells were cultured for an additional 48–72 h.
Whole cell lysate extraction
Cells were harvested by trypsinization, washed with PBS, suspended in lysis buffer containing 10 mM Tris–HCl (pH8), 0.1 M NaCl, 1 mM EGTA, 1 mM EDTA, 0.1% triton X-100, and protease inhibitors (Complete Protease Inhibitor Cocktail tablets, Roche). Extracts were then incubated on ice for 30 min with vortexing every 10 min. Cleared cell lysates were obtained after centrifugation at 16,000 xg for 10 min. SDS-PAGE sample buffer was added to supernatants to achieve final concentrations of 50 mM Tris–HCl with pH 6.8, 1% SDS, 2.5% glycerol, 0.002% bromophenol blue, and 143 mM 2-mercaptoethanol. Samples were then boiled for 2 min at 100°C.
Cells were harvested by trypsinization and washed with PBS. Pellets were fractionated using the NE-PER Nuclear and Cytoplasmic Extraction Kit (Pierce, Rockfield, IL) according to the manufacturer’s protocol. Nuclear and cytoplasmic fractions were prepared in SDS sample buffer and boiled as previously described.
Approximately 20 μg of each lysate or subcellular sample was subjected to 8-12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The proteins were transferred to nitrocellulose by semi-dry transfer at 25 V for 1 h using a Trans-blot SD apparatus (BioRad, Hercules, CA). Membranes were blocked with PBS containing 5% milk and 0.1% Tween-20 (PBST) at room temperature for 1 h and incubated with primary antibodies (1:2000 anti-AIF, 1:2000 anti-PARG, 1:3000 anti-MnSOD, 1:1000 anti-LaminB2, 1:10,000 anti-PAR, 1:10000 anti-beta-actin, 1:2000 anti-caspase3 or 1:2000 anti-caspase-8) in PBST + 5% milk overnight (shaking) at 4°C. Membranes were then washed with PBST three times and incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit or anti-mouse antibody (1:10000) for 1 h. The membranes were washed as described above, and chemiluminescence was initiated using the Supersignal West Pico reagents (Pierce). Immunoblots were then developed on a ChemiDoc XRS gel imaging system (Bio-Rad). For quantification of protein levels for each blot, immunoblots were examined by densitometry with the ChemiDoc imager using Quantity One software. Relative densitometry ratios were then calculated using β-actin as loading controls. The resulting AIF/actin, PARG/actin, or procaspase-8/actin values were then used to quantify relative protein levels.
Cell Viability Assay
Forty-eight hours after cells were transfected with siRNA, cells were treated with 0.5 mM MNNG, an experimental chemotherapeutic agent that methylates DNA  and is known to induce PAR-mediated cell death [12, 33, 46]. Cells were also treated with 20 μM epirubicin (EPI), a dose similar to those used in MDA-MB-231 breast cancer cells in previous reports [47, 48] and consistent with the reported IC50 of this agent in breast cancer cells (55.3 μM) . Other treatments consisted of 10 mM cyclophosphamide (CPA), a dose previously utilized in MDA-MB-231 breast cancer cells , or 0.5 mM cisplatin (DDP), an elevated dose for this agent, but the exposure time utilized (6 hr) was 4 to 12-fold less than normally indicated (24–72 hr). Further, this dose of DDP was consistent with the reported IC50 of this agent in breast cancer cells (0.6 mM) . Twenty hours after treatment, cells were harvested, washed with PBS, and resuspended in annexin-binding buffer (10 mM HEPES, pH 7.4, 140 mM NaCl, 2.5 mM CaCl2,) to approximately 1 X 106 cells/ml. For each 100 μl of cell suspension, 2.5 μl of ApoScreen annexin V-FITC conjugate solution (Southern Biotech) and 1 μl of 100 μg/ml propidium iodide (PI) solution was added. Cells were then incubated at room temperature for 15 min and 400 μl annexin-binding buffer was added. Cell death was then measured by quantifying the percentage of cells that exhibited annexin V-FITC and/or PI fluorescence using a flow cytometer (Accuri, Ann Arbor, MI). Total cell death was quantified by adding the percentage of cells detected in the upper left (PI), upper right (PI + annexin V-FITC), and lower right (annexin V-FITC) quadrants in the FACS dot plots. For pretreatment with Q-VD-OPh, cells were treated with 40 μM Q-VD-OPh 30 min prior to chemotherapeutic treatments. Treatment with Q-VD-OPh continued during all treatments, and until FACS analyses were performed.
Caspase Activity Assay
Wild type and PARG null TS cells were cultured and exposed to 25 J/m2 UV-C using a low-pressure Hg lamp (model G30T8, Sylvania, Danvers, MA). 12 h after UV exposure, 1 X 105 cells were seeded in 96-well opaque black plate and subjected to Apo-ONE Homogeneous Caspase-3/7, Caspase-8, and Caspase-9 Assays (Promega) according the manufacturer’s specifications. Fluorescence was analyzed using a Bio-Tek (Winooski, VT) Synergy HT plate reader at an excitation wavelength of 485 nm and at an emission wavelength of 527 nm. Negative controls were provided by untreated cells and cells pretreated with 40 μM Q-VD-OPh.
All error bars for calculated cell death, caspase activity, and protein levels represented the standard error of the mean (SEM). Statistical analyses were accomplished by one-way analysis of variance (ANOVA) and unpaired Student’s t-test.