The COMBAT approach based on empirical Bayes frameworks was applied to remove batch effects across the two batches of microarray experiments (18). Human tissue samples Human tissues used for microarray analysis were collected following written informed consent and clinically annotated according to established protocols and approved by the appropriate Institutional Review Boards (IRB) at the Moffitt Malignancy Center and Vanderbilt University or college as previously explained (“type”:”entrez-geo”,”attrs”:”text”:”GSE17536″,”term_id”:”17536″GSE17536 and “type”:”entrez-geo”,”attrs”:”text”:”GSE17537″,”term_id”:”17537″GSE17537) (15). RNA preparation and analysis Total RNA from cells E2F1 or tissues was isolated using QIAGEN kits (QIAGEN, Valencia, CA) and DNase-I treated, quantified by Nanodrop-1000 (Thermo Scientific, Rockford, IL) and assessed for quality on an Agilent Bioanalyzer as previously described (15). of microarray experiments (18). Human tissue samples Human tissues used for microarray analysis were collected following written informed consent and clinically annotated according to established protocols and approved by the appropriate Institutional Review Boards (IRB) at the Moffitt Malignancy Center and Vanderbilt University or college as previously explained (“type”:”entrez-geo”,”attrs”:”text”:”GSE17536″,”term_id”:”17536″GSE17536 and “type”:”entrez-geo”,”attrs”:”text”:”GSE17537″,”term_id”:”17537″GSE17537) (15). RNA preparation and analysis Total RNA from cells or tissues was isolated using QIAGEN kits (QIAGEN, Valencia, CA) RG7112 and DNase-I treated, quantified by Nanodrop-1000 (Thermo Scientific, Rockford, IL) and assessed for quality on an Agilent Bioanalyzer as previously explained (15). Chromosome Immunoprecipitation (ChIP) studies were conducted using mouse anti-NFATc1 specific antibody from Santa Cruz Biotechnology (Santa Cruz, California) and an kit from Millipore (Billerica, MA), according to the manufacturers instructions. Quantitative RT-PCR (qPCR) RG7112 was performed as explained elsewhere (19), gene specific primers are outlined in Supplementary Furniture 2C3. Cell culture The MC-38 mouse adenocarcinoma cell collection and its derivatives were provided by Dr. Robert Coffey are explained elsewhere (15). HCT116 and HT29 colon cancer cell lines were obtained from American Type Culture Collection (Manassas, Virginia). All cell lines were managed at low passage as monolayers in RPMI-1640 medium (Gibco Life Technologies, Rockville, MD) supplemented with 10% fetal bovine serum (Atlanta Biologicals, Lawrenceville, GA) 500U/ml Penicillin G, 500 g/ml Streptomycin (Gibco Life Technologies Inc., Rockville, MD) and L-glutamine (Gibco Life Technologies Inc., Rockville, MD). FK506 (Sigma) was used at 20ng/mL. Integrity of human cell lines used in this study was tested by RNAseq analysis in May 2013. Cytoplasmic and nuclear extracts were prepared using Nuclear Extract kit (Active Motif, Carlsbad, CA), according to manufacturers instructions. Immunoblots Cells were harvested in RIPA lysis buffer (50mM Tris pH7.5, 150mM NaCl, 1% NP-40, 0.5% Na-deoxy Cholate, 0.1%SDS) containing a cocktail of protease inhibitors (Roche Diagnostics, Indianapolis, IN), with a brief sonication. Samples were mixed with LDS buffer made up of DTT (Invitrogen, Carlsbad, CA), and fractionated on 4C12% NuPAGE gels in MOPS-SDS buffer (Invitrogen, Carlsbad, CA). Antibodies against NFATc1-c4 and PARP1/2 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA) -Actin from Sigma Chemical (St. Louis, Mo) and -Tubulin from Abcam Scientific (Cambridge, Mass.). Ramos cell (Burkitts lymphoma, B lymphocytes) lysate (Santacruz Biotechnology) was used as positive control. Invasion assays Invasion assays were conducted using both Boyden chambers as explained elsewhere (15) as well as the xCELLigence system from Roche Diagnostics (50) (observe also Supplementary Methods). Overexpression and inhibition of NFATc1 RNAi studies were performed as previously explained using NFATc1 specific ON-target plus SMART pool siRNA or ON-target plus Non-targeting Pool (Thermo Scientific; Rockford, IL,) Specific si-RNA sequences are given in Supplementary Table 4. For preparing stable over-expressing cells, mouse wild-type NFATc1 (Addgene plasmid 11101) (20) was cloned into vector pGP-Lenti3 (GenBank Accession no. “type”:”entrez-nucleotide”,”attrs”:”text”:”JX861384″,”term_id”:”413968724″,”term_text”:”JX861384″JX861384) between unique XbaI and BamHI sites. Puromycin and GFP expression were used as selection markers for creation of stable cell lines. For gene knockdown experiments, NFATc1 specific GIPZ lentiviral shRNAmir RG7112 (V3LMM_418820) (Thermoscientific) was selected for experimental use. Briefly, MC38 cells were electroporated using NEON (Invitrogen). Puromycin and GFP expression were used as selection markers for creation of stable cell lines. For HT29 studies, cells were transiently co-transfected with pEF6-NFATc1 and pMaxGFP vectors, circulation sorted to enrich for highly expressing GFP-positive cells and confirmed NFATc1 overexpression by Western blot. Animal studies All animal studies were approved by the.
