Gadd45a

Total RNA extracted from tumors, normal tissues, cell lines, xenograft tumors, and explants were subjected to qRT-PCR analysis and western blot as described previously (13 (link)). Supplementary Table S1 lists primer sequences for all genes studied in the present study. Antibodies against β-actin (A3854) and Aurora kinase A (T6199) were purchased from Sigma Aldrich. Antibodies against FoxM1 (sc-502), XRCC3 (sc-271714), EXO-1 (sc-19941) and GADD45-A (sc-797) were purchased from Santa Cruz. Antibody against PLK (#627701) was purchased from BioLegend and against NOX4 (NB110-58849) was purchased from Novus. Calnexin antibody was kindly provided by Dr. Hima Bansal, UTHSCSA, San Antonio, TX). Other antibodies including SKP2 (#4358), cyclin D1 (#2978), E2 (#4132), CDK2 (#2546), CDK4 (#12790), p-JNK (#9251), total JNK (#9252) and NFκB p65 (#8242) were purchased from Cell Signaling. RAD51 (#70005) antibody was purchased from BioAcademia.

Western blot analysis was performed by standard methods as previously described (31 (link)). Relevant commercially available antibodies were used as indicated per experiment: p53, p21, GADD45a (Santa Cruz); MDM2, Nup107 (Abcam). Horseradish peroxidase (HRP)-conjugated secondary antibodies (Santa Cruz) were detected by ECL (PerkinElmer).
In PD studies, tumor tissues were frozen until analysis. They were then lysed after mechanical dissociation using a bead beating Precellys homogenizer 24 (Ozyme) with a Cryolys cooling system in ice-cold lysis buffer: 10 mM Tris (pH 7.5), 100 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton, 1 mM NaF, 20 mM Na4P2O, 1 mM activated Na3VO4, 10% glycerol supplemented with protease inhibitor tablets. Lysates were incubated for 2 hours at 4°C, clarified by centrifugation and supernatants were collected, aliquoted and stored at −80°C until analysis. Total protein concentrations were determined with the BCA protein assay kit (Thermo scientific).
For Mesoscale Discovery assays, protein analysis was performed after appropriate dilutions with following kits: MDM2 (K152FID), p21 (N45ZA-1), p53, Cleaved Caspase 3 and cleaved PARP (K15102D-1) with a detection on Sector Imager 2400. Results were normalized with total protein concentration.