Cell culture, transfection, and treatment
U2OS osteosarcoma cells, HeLa cells, and 293T cells were purchased from ATCC and cultured in DMEM (Sigma-Aldrich, Schnelldorf, Germany) supplemented with 10% FBS (Sigma-Aldrich) and 1% antibiotics (penicillin) (Gibco; Thermo Fisher Scientific, Waltham, MA) at 37°C and 5% CO2. DR-GFP U2OS cells were kindly provided by Prof. Thomas Hellday and Dr. Oliver Mortusewicz and cultured in DMEM with 10% FBS, antibiotics and 1 µg/ml puromycin (Gibco). For siRNA-mediated knockdown, cells were transfected with 5 nM predesigned Silence Select siRNAs or control siRNA (Ambion/Life Technologies) using INTERFERin (Polyplus Transfection, Illkirch, France). For plasmid transfection, jetPEI (Polyplus Transfection) was used. Chemotherapeutic treatment was performed using hydroxyurea (HU; Sigma-Aldrich) dissolved in H2O, and X-rays were generated using a CellRad X (Faxitron, USA).
Small interfering RNA (siRNA)
siRNA-mediated knockdown was achieved using INTERFERin (Polyplus Transfection) following the manufacturer’s instructions. Predesigned Silence Select siRNAs or control siRNA (Ambion/Life Technologies) were used. Each siRNA was used as a pool of three target-specific siRNAs: non-targeting, Mission SIC-001 (Sigma-Aldrich); MED1, #s10889 (5′-GCUGGUCCCUUGGAUAAGAtt-3′, 3′-UCUUAUCCAAGGGACCAGCat-5′), #s10890 (5′-GACCAGUCCUUGUCUAUGAtt-3′, 3′-UCAUAGACAAGGACUGGUCtg-5′), and #s10891 (5′-CCAGUACAGGUGGUGGAUCUAAtt-3′, 3′-UUAGAUCCACCUGUACUGGta-5′); and BRCA1, #s457 (5′-GGGAUACCAUGCAACAUAAtt-3′, 3′-UUAUGUUGCAUGGUAUCCCtc-5′), #s458 (5′-CAGCUACCCUUCCAUCAUAtt-3′, 3′-UAUGAUGGAAGGGUAGCUGtt-5′), and #s459 (5′-CAUGCAACAUAACCUGAUAtt-3′, 3′-UAUCAGGUUAUGUUGCAUGgt-5′). siRNAs were transfected with INTERFERin (Polyplus Transfection) at a final concentration of 5 nM.
Plasmid
The pCMV-I SceI plasmid and pcDNA-RNaseH1 vector were kindly provided by Prof. Thomas Hellday and Dr. Oliver Mortusewicz. The pcDNA3, pGEX 4T-1, and pRL Renilla CMV-Luc control reporter vectors were purchased from Invitrogen (Camarillo, CA), GE Healthcare (Buckinghamshire, UK), and Promega (Madison, WI), respectively. The pGEX 4T-1 BRCT, GAL4, GAL4-BRCT, and pcDNA-MED1 vectors and reporter constructs (17M8-AdMLP-luc) were described previously by Hiraike et al. (2010)(28).
Antibodies
Used primary and secondary antibodies are listed in Supplemental Table 1.
Immunoblotting
Total cell extracts were obtained using RIPA buffer (50mM Tris-HCl pH 8.0, 150 mM NaCl, 0.5% w/v sodium deoxycholate, 0.1% w/v sodium dodecyl sulfate, 1% w/v NP-40) with complete, EDTA-free (Roche, Basel, Switzerland), and Halt Phosphatase inhibitor cocktail (Thermo Scientific). Sample buffer (1 M Tris-HCl pH 6.8, 40% w/v glycerol, 20% w/v 2-Mercaptoethanol, 8% w/v sodium dodecyl sulfate, 0.02% w/v bromophenol blue) was added to the extracts. Equivalent amounts of lysate protein (10 µg) were used in Mini-PROTEAN TGX Precast Protein Gels (Bio-Rad) and electrophoretically transferred onto Trans-Blot Turbo Transfer Packs (Bio-Rad) using the Trans-Blot Turbo Transfer System (Bio-Rad). Proteins transferred onto membranes (Bio-Rad) were probed with appropriate primary and secondary antibodies. Target protein expression was detected using ECL Plus WB detection reagents (GE Healthcare) using Image Quant LAS 4000 mini (GE Healthcare).
Immunoprecipitation
The formation of a MED1-BRCA1 complex in U2OS cells was analyzed by IP. Whole-cell extracts of U2OS cells using RIPA buffer were used for IP with anti-BRCA1 and anti-MED1 antibodies or preimmune IgG. The immunoprecipitate was subjected to 30 ml of protein G Sepharose 4 Fast Flow (GE Healthcare) and subsequently immunoblotted with anti-BRCA1, anti-MED1, or anti-IgG antibodies.
Immunofluorescence
Cells plated on coverslips were fixed for 10 min at room temperature with fixative (3% paraformaldehyde [PFA], 0.1% Triton X-100, 1⋅ PBS). For RPA staining, pre-extraction was performed for 5 min with ice-cold 0.3% TritonX-100 in CSK buffer (100 mM NaCl, 300 mM sucrose, 3 mM MgCl2, 10 mM PIPES, 1 mM ethylene glycol-bis (β-aminoethyl ether)-N,N,N0,N0-tetraacetic acid). After fixation, cells were rinsed briefly with 0.05% Tween 20 in PBS twice, and then permeabilized for 10 min with 0.3% Triton X-100 in PBS. After blocking for 40 min with PBS+3% BSA or skim milk in PBS, cells were incubated with primary antibodies diluted in PBS+3% BSA or skim milk in PBS at 4°C overnight in a wet chamber. Cells were again rinsed twice with 0.05% Tween 20 in PBS and then permeabilized for 10 min with 0.3% Triton X-100 in PBS. Cells were then incubated with secondary antibodies diluted in PBS+3% BSA at room temperature in the dark for 1 h. After washing with 0.05% Tween 20 in PBS, nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI) for 5 min, washed with 0.05% Tween 20 in PBS, and permeabilized with 0.3% Triton X-100 in PBS. After rinsing with D2W, slides were mounted with ProLong Gold (Life Technologies) and visualized using a confocal microscope LSM780 (Carl Zeiss, Le Pecq, France). For foci quantification, cells were fixated in 96 well plates (BD Falcon) using the same protocol, and images were taken using BZ-X710 (Keyence, Osaka, Japan) and analyzed using Image J software (NIH, Bethesda, Maryland, USA). More than 400 cells were counted. Error bars represent SEM from three independent experiments.
GST-pull down assay
Glutathione-S-transferase fusion proteins or GST alone were expressed in Escherichia coli and immobilized on glutathione Sepharose 4 B beads (GE Healthcare). GST proteins were incubated with whole-cell extracts of U2OS cells. Unbound proteins were removed, and bound proteins were eluted and analyzed by SDS-PAGE.
Luciferase assay
293T cells were seeded (6.0 × 104/well) in 12-well plates and transfected with the indicated expression vectors using jetPEI (Polyplus transfection, Ilkirch, France). Luciferase assays were performed using the dual luciferase reporter assay system (Promega). Expression vectors were co-transfected with reporter plasmid (17M8-AdMLP-Luc). As an internal transfection control, phRL CMV-Renilla vector (Promega) was also transfected in all experiments. After 24-48 h of transfection, cells were harvested, washed with PBS, and fixed following manufacturer’s recommendations. Luminescence was measured using an LB96V luminometer (Berthold Technologies, Bad Wildbad, Germany). Individual transfections, each consisting of triplicate wells, were repeated at least three times.
Colony formation assay
HeLa cells (8.0 × 104/well) were seeded in 6-well plates. After 24 h of incubation, endogenous MED1 was depleted with siRNA-mediated knockdown. Two days after siRNA transfection, cells were collected by trypsinization, treated with different doses of radiation, and reseeded into 6-well plates at 1 000 cells/well. Alternatively, cells were reseeded and cultured in various concentrations of cisplatin. The medium and cisplatin were replaced every three days. After 7 days of incubation, the cells were fixed with 100% methanol for 2 h and stained with Giemsa (Wako, Osaka, Japan). The dishes were gently washed with water and air-dried. Colonies with more than 50 cells were counted. Results are presented as the mean ± SEM from three independent experiments.
HR and NHEJ assays
HR and NHEJ assays were performed in DR-GFP USOS and EJ5-GFP U2OS cells, respectively. Cells were seeded in 12 well plates (5.0 x 104 cells/well). Twenty-four hours after transfection of siRNA against endogenous MED1 or BRCA1, or non-targeting siRNA, cells were transfected with plasmids expressing I-SceI endonuclease or an empty vector as a control. After 48 h, cells were harvested, fixed with 4% PFA, and subjected to FACS analysis to determine the efficiency of HR or C-NHEJ repair induced by I-SceI digestion, which reconstituted a functional GFP gene. GFP signals were quantitated on a FACS Navios flow cytometer (BD FACSCalibur HG, BD Bioscience, Franklin Lakes, NJ, USA) and analyzed with Cell Quest Pro software v 3.1 (BD Bioscience). For each experiment, 20 000 cells were scored per treatment group, and the frequency of recombination events was calculated from the number of GFP-positive cells divided by the number of cells analyzed. Data are presented as the mean ± SEM from three independent experiments with p-values determined by Student’s t-test.
Alkaline single-cell gel electrophoresis comet assay
U2OS cells were seeded in 6-well plates (1.0 x 105 cells/well). Twenty-four hours later, cells were transfected with non-targeting siRNA or MED1 pool siRNAs using INTERFERin (Polyplus Transfection). After another 24 h, cells were treated with X-rays and incubated for 24 h before harvesting. After washing with PBS, cells were re-suspended in PBS at a concentration of ~ 1.0 × 106 cells/ml. DNA damage was evaluated using the Comet Assay Kit (4250-050-K, Trevigen) following manufacturer’s instructions. Electrophoresis was run at 300 mA, 21 V for 30 min in electrophoresis buffer using the Comet Assay Electrophoresis System II (Trevigen). Slides were washed in neutralization buffer (0.4 M Tris-HCl pH 7.5) and counterstained with SYBR Gold (diluted 1:1000 in PBS) (Invitrogen). Images were acquired using a confocal microscope LSM780 (Carl Zeiss), and comet tail moment was estimated using CometScore software. At least 100 comets per sample were analyzed.
Cell cycle analysis using double thymidine block
U2OS cells were seeded (8.0 x 104 cells/well) in 6-well plates and transfected with indicated siRNAs. After 24 h of incubation, cells were reseeded in 6-well plates (2.0 x 105 cells/well). For cell cycle synchronization, cells were arrested twice at the G1/S boundary using a double incubation in the presence of 2.5 mM thymidine for 15-18 h, followed by a 9-h interval of growth without the drug. Cells were released from the cell cycle blocks and harvested using trypsin at the indicated times. Cells were stained in the dark with 50 µg/mL propidium iodide (PI) (Sigma-Aldrich) at 4°C for 30 min. Cell cycle distribution was analyzed by flow cytometry on an Epics XL instrument (Beckman Coulter, Brea, CA, USA) using Cell Quest Pro software v 3.1 (BD Bioscience, Franklin Lakes, NJ, USA). The data are presented as the mean ± SEM from three independent experiments.
Statistical analysis
Statistical analyses were performed using GraphPad Prism 7 software (GraphPad Software, San Diego, CA). Results are shown as mean ± standard error of the mean. Data were analyzed using Student’s t-test for paired comparisons. Statistical significance was set at P < 0.05.