Nu serum 4

Dissociated human and mouse tumour cells and 4T1 cells were cultured in RPMI 1640 supplemented with GlutaMAX (Gibco, 61870010), 10% foetal bovine serum (FBS, Eurobio Scientific, CVFSVF00–01). HT-1080 cells were cultured in Dulbecco’s Modified Eagle Medium GlutaMAX (DMEM, Gibco, 61965059) supplemented with 10% FBS (Gibco, 10270–106) and penicillin/streptomycin (BioWhittaker/Lonza, DE17–602E). FC1242 and FC1245 murine pancreatic cancer cells, 4a cells and human pancreatic hMIA-2D cells were a generous gift from the Tuveson laboratory (CSHL) and were cultured in DMEM supplemented with 10% FBS and penicillin/streptomycin. Primary human pancreatic PDAC090T, PDAC053T, PDAC211T and PDAC030T cells were grown in serum-free ductal medium: DMEM/F12 supplemented with 0.61g/500μL nicotinamide (Sigma-Aldrich, 3376), 2.50g/ 500μL glucose (Sigma-Aldrich, G6152), 1:200 ITS+ (Corning, 354352), 1:20 Nu-serum IV (Corning, 355104), 100 ng/μL. cholera toxin, 1 μM dexamethasone (Sigma-Aldrich, D4902), 50 nM 3,3’,5-triiodo-L-thyronine (Sigma-Aldrich, T6397) and penicillin/streptomycin.

The DU145 (human prostate adenocarcinoma, radio-resistant, p53 deficient, derived from a brain metastasis), TRAMP-C2 (prostate adenocarcinoma, radio-resistant, wild-type p53, derived from 32-week old TRAMP mice) and human kidney embryonic HEK-293 cell lines were obtained from the American Type Culture Collection (ATCC, Rockville, MD). DU145 cells were grown in RPMI-1640 (Hyclone, Waltham, MA) supplemented with 10% fetal bovine serum (FBS) (Hyclone), and 100-units/ml penicillin supplemented with 1 mg/ml streptomycin (Hyclone). TRAMP-C2 cells were grown in Dulbecco’s modified Eagle’s medium (Hyclone) supplemented with 5% FBS (Hyclone), 5% Nu-Serum IV (Corning, Corning, NY), 5 μg/ml bovine insulin (Sigma Aldrich, St. Louis, MO), 10 nM dehydroisoandrosterone 90% (Sigma Aldrich), and 100 units/ml penicillin supplemented with 1 mg/ml streptomycin (Hyclone). The HEK-293 cells were grown in Dulbecco’s modified Eagle’s medium (Hyclone) supplemented with 10% FBS (Hyclone). All cells were grown at 37 °C, in a 5% CO₂ in 95% atmosphere incubator.

The simian virus 40 large T antigen-transformed human brain microvascular endothelial cells (HBMEC) were cultured as previously described^{56 (link)–58} . Briefly, HBMECs were cultured in RPMI-1640 medium (Gibco Life Technologies, Karlsruhe, Germany) supplemented with 10% fetal calf serum (FCS, Gibco Life Technologies), 10% Nu serum^® IV (Corning, NY, USA 10%; Becton Dickinson), 1% sodium pyruvate (1 mM), 1% L-glutamine (2 mM), 1% non-essential amino acids (all purchased from GE Healthcare, Little Chalfont, UK), 5 U ml⁻¹ heparin (Biochrom, Berline, Germany) and 30 µg mL⁻¹ endothelial cell growth supplement (ECGS, CellSystems, Troisdorf, Germany). Cultures were incubated in a humid atmosphere at 37 °C with 5% CO₂. Cells between the 10th and 25th passages were used for apoptosis analysis. HBMEC were cultured in T25 flasks (Corning Costar Corporation, Cambridge, MA, USA). The embryonic kidney cell line HEK293T and the hepatocellular carcinoma cell line HepG2 were purchased from the American Type Culture Collection, ATCC^® CRL-3216^™ and ATCC^® HB-8065^™, respectively. HEK293T and HepG2 were routinely cultured in Dulbecco’s modified Eagle’s medium (DMEM) + GlutaMAX^™-I (Gibco Life Technologies, Karlsruhe, Germany) supplemented with 10% FCS.

TRAMP-C2 cells, an epithelial line isolated from tumor burdened TRAMP prostates, were maintained in High-Glucose DMEM (Hyclone) supplemented with 0.005 mg/ml bovine insulin (Sigma), 10nM dehydroisoandrosterone (Sigma), 5% fetal bovine serum (Atlanta), 5% Nu-Serum IV (Corning), and 1% penicillin/streptomycin (Life Technologies). One day prior to transfection, cells were plated at 4 x 10⁵ cells/well in 6-wells plates. 50nM Dharmacon ON-TARGETplus SMARTpools for non-targeting control #1 (D-001810-01-05), siRunx1 (L-048982-00), or siRunx2 (L-012665-00-0005) were transfected into cells with Lipofectamine 2000 (Life Technologies) as per manufacturers recommended protocol.

4T1 cells were cultured in DMEM high glucose (HyClone, GE Healthcare, South Logan, UT) with 10% of fetal bovine serum (FBS; Peak Serum, Wellington, CO) and 1% antibiotic/antimycotic (HyClone, GE Healthcare, South Logan, UT). A TPSA23 cell line was previously constructed from the murine prostate adenocarcinoma cell line to secrete human PSA, as described^{16 (link)}. TPSA23 cells were cultured in DMEM high glucose, 5% FBS, 5% Nu-Serum IV (Corning, Corning, NY), 10 nM dehydroisoandrosterone (Sigma-Aldrich, St. Louis, MO), 5ug/ml bovine insulin (Sigma- Aldrich, St. Louis, MO), 1% antibiotic/antimycotic.