A375 melanoma cells and Human Embryonic Kidney 293T (HEK293T) cells were obtained from ATCC (Manassas, VA). MRA melanoma cells, low-passage cultures derived from metastatic melanoma patients, were provided by Dr Mark R. Albertini. Cells were propagated in DMEM medium supplemented with 10% tet-system-approved fetal bovine serum (Clontech, Mountain View, CA) and 1% penicillin–streptomycin.
Transfections and lentiviral transduction
Tetracycline-responsive TRIPZ shRNAmir, MAGE-C2 or scrambled, and the Trans-Lentiviral packaging system were purchased from Open Biosystems (ThermoFisher Scientific, Huntsville, AL). Lentiviruses were produced, as per manufacturer’s recommendations, and concentrated with Lentivirus Concentrator (Clontech). A375 and MRA cells were transduced with concentrated lentiviruses expressing scrambled or MAGE-C2 shRNA and selected with puromycin. Transduction efficiency, determined by expression of TurboRFP, which is also encoded by the lentivirus vector, was ~70% in A375 cells and ~90–100% in MRA-13 cells. MRA-13 cultures were used as bulk cultures, whereas single-cell clones of transduced A375 cells were selected on the basis of inducible RFP expression. MAGE-C2 knockdown was confirmed by immunoblotting. HEK293T cells were transfected with vector, MAGE-C2, or mutant MAGE-C2 by the calcium phosphate method and ATM 472 siRNA (Dharmacon, Lafayette, CO) by Lipofectamine 2000 (Life Technologies, Grand Island, NY).
Immunoprecipitations and immunoblotting
A375 and MRA cells, transduced with scrambled or MAGE-C2 shRNA, were treated with doxycycline and collected after 72 hours. HEK293T cells were transfected with empty plasmid, MAGE-C2, or mutant MAGE-C2-expression plasmid. Protein lysates were immunoprecipitated with ATM antibody (Abcam, Cambridge, MA). The expression of MAGE-C2, Actin (Santa Cruz Biotechnology, Santa Cruz, CA), phospho-KAP1, KAP1 (Novus Biologicals, Littleton, CO), M2 FLAG (Sigma-Aldrich, St Louis, MI), and ATM (Abcam) was analyzed by immunoblotting. Density analysis was performed using ImageJ software (National Institutes of Health, Bethesda, MD) and was calculated for loading of the controls as actin or total protein expression for phosphorylated proteins. Density of the bands is shown as numbers under the blots.
Cytostaining and TUNEL assay
A375 cells, transduced with lentivirus encoding scrambled or MAGE-C2 shRNA and treated with 0.5 μgml−1 doxycycline for 72 hours, were stained with MAGE-C2 antibody (Santa Cruz Biotechnology) or phospho histone H2A.X (Cell Signaling, Danvers, MA) and FITC-labeled secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA). MRA-13 and A375 melanoma cells, transduced with lentivirus encoding scrambled or MAGE-C2 shRNA and treated with doxycycline for 96 hours, were stained for TUNEL using the FITC-labeled DeadEnd Fluorometric TUNEL System (Promega, Madison, WI). These cells were co-stained with MAGE-C2 (Santa Cruz Biotechnology). Mouse MRA-13 tumor xenograft sections were also stained with TUNEL. Quantitative analysis of TUNEL stain and fluorescent MAGE-C2 or phospho-H2AX was carried out using NIS Elements software (Nikon, Melville, NY).
Athymic nude mice xenografts
MRA-13 cells, transduced with lentivirus encoding tetracycline-responsive scrambled or MAGE-C2 shRNA and selected with puromycin, were used for xenograft studies. Transduction efficiency was 90–100% as detected by RFP expression after treatment with doxycycline. Four million MAGE-C2 or scrambled shRNA tumor cells were injected subcutaneously into 4-week-old athymic nude mice (Harlan Laboratories, Indianapolis, IN). A week later, half of the mice were given 2mgml−1 doxycycline in drinking water, with seven mice per group. The xenograft experiment was terminated 6 weeks later or when tumors were 1,400mm3 in volume, as per protocol.
Tumors collected from athymic nude mice xenografts were fixed in formalin and embedded in paraffin. Paraffin sections were stained with MAGE-C2 antibody and developed by 3,3-diaminobenzidine (Sigma-Aldrich, St. Louis, MO).
ATM kinase inhibitor (KU55933) treatment
HEK293T cells, transfected with empty vector or MAGE-C2 expression plasmid, were treated with KU55933 ATM kinase inhibitor (Tocris Biosciences, Ellisville, MO) for 24 hours. Cells were collected and lysed as mentioned before. The expression of MAGE-C2, phospho-KAP1, KAP1, phospho p53, p53, and actin was evaluated by immunoblotting.
DNA damage and repair assay
U-2 OS cells, containing an integrated modified gene for GFP, were used to measure DNA damage repair (Pierce et al., 1999
). Cells were co-transfected with I-SceI pCbASce plasmid and empty vector or MAGE-C2. After 48 hours, cells were immunostained for MAGE-C2 expression (red). The expression of MAGE and GFP was enumerated and compared, including measurement of staining intensity using NIS Elements software.