Human MDA-MB-231 breast and A2780 ovarian adenocarcinoma cells (ATCC, Manassas, VA) were cultured in RPMI medium containing 10% (v/v) fetal bovine serum, penicillin and streptomycin (Invitrogen, Carlsbad, CA). HeLa S3 (S3) cells and derivatives, HEK293T cells and Linx cells (Open Biosystems, Thermo Fisher Scientific, Huntsville, AL) where cultured in DMEM containing 10% (v/v) calf serum (Invitrogen) and antibiotics.
Cisplatin (cis-diamminedichloro-platinum) powder (Sigma-Aldrich, St Louis, MO) was prepared freshly in cell culture medium. Nu7026 (Sigma) was dissolved in DMSO and stored at -20 C.
Mouse monoclonal antibodies used were anti-DNA-PKcs 18-2, anti-Ku86 111 and anti-Ku70 N3H10 (Neomarkers/Labvision, Thermo Fisher Scientific), anti-FLAG M2 (Sigma-Aldrich), anti-SSRP1 and anti-SPT16 (Biolegend, San Diego, CA), anti-phospho(ser139)-H2AX clone JBW301 (Abcam, Cambridge, MA), anti-Poly (ADP ribose) Polymerase-1 (PARP-1, BD Biosciences, San Jose, CA). Rabbit antibodies were anti-RHA, anti-WRN (Santa Cruz Biotechnology, Santa Cruz, CA), anti-H2A (Upstate, Millipore, Billerica, MA), anti-phospho(ser139)-H2AX (Abcam), anti-cleaved caspase-3 (Cell Signaling Technology, Danvers, MA) and anti-DNA-PKcs phospho-serine 2056 (a kind gift from Dr. B. Chen) .
Cells grown on glass coverslips were fixed with 3.7% paraformaldehyde (PFA) and permeabilized with methanol for 10 minutes. The cells were incubated with primary antibodies at a 1/300 dilution for 1 hr at 37C, rinsed with PBS and incubated for 30 min at 37C with secondary antibodies conjugated to Alexa Fluor-488 or Alexa Fluor-594 fluorochromes (Molecular Probes, Invitrogen), at a 1/300 dilution. DNA was visualized by DAPI (Sigma-Aldrich). Fluorochromes were visualized with an Axioskop II microscope and imaged with AxioVision 4.5 software (Zeiss, Jena, Germany).
Assays of DNA damage
Laser-induced DNA DSBs were generated using a P.A.L.M. MicroBeam laser microdissection system (Zeiss) at λ = 337 nm as previously described [48, 49]. Cells were grown on coverslips for 24 hours in media containing 10 uM BrdU (Sigma-Aldrich) prior to laser treatment. After laser stripe generation, cells were incubated at 37°C and fixed 60 min later for immunofluorescence. DNA damage-induced foci were generated either by γ-irradiation or cisplatin and visualized by γH2AX immunofluorescence .
Plasmids and transfection
For DNA-PKcs silencing, retroviruses were produced by transfecting retroviral pSM2c expression vector (Open Biosystems) containing a puromycin resistance gene and a control shRNA (5'TCTCGCTTGGGCGAGAGTAAG) or a shRNA to DNA-PKcs (5'GGAGCTTACATGCTAATGTAT) into the Linx packaging cell line. On day three, virus-containing supernatants were added to MDA-MB-231 and A2780 cells and incubated in 5 μg/ml polybrene. For knock down of FACT, small hairpin sequences specific to SSRP1 (5'CACCACAGTACTGCGTCTGTT) were cloned into the pcDNA6.2 vector (Invitrogen) containing a blastocidin resistance gene, according to manufacturer instructions. A control shRNA sequence (5'GTCTCCACGCGCACTACATTT) was used to generate a non-silencing control plasmid. Transfection of HEK293T, MDA-MB-231 and A2780 cells was assisted by FuGENE HD (Roche, Indianapolis, IN). Selection with puromycin or blastocidin started 48 hrs after transfection. After 10 days resistant colonies where expanded and protein knock down determined by immunoblotting and immunofluorescence.
Tandem affinity purifications
HeLa S3 cells expressing Ku86 fused to Flag and HA tags (S3-Ku86-Flag/HA) were treated as indicated. Sub-cellular fractions were prepared as described . Briefly, cells were incubated in hypotonic buffer (10 mM Tris-HCl [pH 8], 10 mM KCl, 1.5 mM MgCl2) on ice for 10 min and homogenized by tight dounce. Nuclei were collected by centrifugation at 2000 g for 15 min at 4°C and extracted with 40 mM Tris-HCl [pH 8], 200 mM NaCl, 10% glycerol, 2 mM EDTA, 0.5% NP40 and a 1 × protease inhibitor mix for 45 min at 4°C. The insoluble material (chromatin) was pelleted at 15,000 g for 30 min at 4°C, and the supernatant called "nuclear extract" (NEX). NEX or chromatin were further treated through two sequential FLAG and HA immunoprecipitations as previously described .
Chromatin pellets obtained as described above were washed in 20 mM Tris-HCL pH 7.5, 100 mM KCl, 2 mM MgCl2, and 1 mM CaCl2 and incubated at room temperature in 0.05 U/μl micrococcal nuclease (MNase, Nuclease S7, Roche) for 15 min, pelleted and the supernatant was collected.
Samples were separated on NuPAGE 4-12% gels (Invitrogen), analyzed by immunoblotting with the indicated antibodies and visualized with Supersignal chemi-luminescent reagents (Pierce, Thermo Fisher Scientific) and a luminescent image analyzer LAS-3000mini (Fujifilm, Edison, NJ). When indicated relative amounts of proteins were compared using ImageJ software.
Cytotoxicity was assessed by the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfenyl)-2H-tetrazolium, inner salt (MTS) assay using the CellTiter 96 Aqueous One Solution Proliferation Assay (Promega, Madison, WI), according to the manufacturer's protocol. Cells were seeded in 96-well plates at a density of 5000 cells/well. After overnight incubation, cisplatin was added at the concentrations indicated. The absorbance of each well was measured at 490 nm. Values for control cells were considered as 100% viability. The dose-response curves were plotted as a percentage absorbance of control cells. The half maximal inhibition (IC50) value was calculated from the percent inhibition curve generated using Excel XLfit software (Microsoft, Redmond, WA).
The Cell Death Detection ELISA Assay (Roche) was used to analyze apoptosis and necrosis in response to cisplatin. The assay is a sandwich-enzyme-immunoassay using antibodies directed against DNA and histones and allowing quantification of nucleosomes. Nucleosomes were quantified either in the cell culture supernatant (necrosis) or in cell lysates (apoptosis). The assay was performed as recommended by the supplier.
For the MTS and Cell Death Detection ELISA assays, all values were expressed as mean ± s.e.m. Differences between groups were tested for statistical significance using Student's paired t-test. P < 0.05 represented a significant difference.