Dmem f 12 medium

T47D, BT474, MDA-MB-231, ZR-75-1, and MCF7 breast cancer cell lines and an immortalized normal-like breast cell line 184A1 were obtained as a gift from the laboratory of Dr. Dennis J. Slamon (UCLA, Los Angeles, CA). The cell lines were authenticated by DNA short tandem repeat profiling using the Promega GenePrint 10 system, and analysis was done using the GeneMarker HID software and the ATCC database (Table S3). Cells were tested for mycoplasma and were made mycoplasma-free using EZKill mycoplasma removal reagent (HiMedia). T47D, BT474, MDA-MB-231, MCF7, and ZR-75-1 cells were cultured as described previously (19 (link), 36 (link)). The immortalized normal-like 184A1 cell line was cultured in DMEM/F-12 medium (HiMedia) supplemented with 28.18 IU of insulin, 20 ng/ml EGF, and 500 ng/ml hydrocortisone. Basal medium was supplemented with 10% (v/v) fetal bovine serum (Gibco), 2.5 mg/ml Amphotericin-B (Abbott), and 1.25 μl/ml gentamycin (Abbott). All of the cells were cultured at 37 °C in a 5% CO₂ incubator. The ER/PR/Her2 receptor status of all of the cells was validated by RT-PCR and expression array analysis (Table 1).

Acrylamide, bovine serum albumin (BSA), bromophenol blue, 7,12-dimethylbenz[a]anthracene (DMBA), hydroxyurea, 2-mercaptoethanol, sodium dodecyl sulphate (SDS) N,N,N′,N′ - tetramethylene diamine (TEMED) and Trizol were purchased from Sigma Chemical Company, St. Louis, MO, USA. Astaxanthin was procured from Bio-Real, Sweden. DMEM-F12 medium, antibiotic solution consisting of penicillin and streptomycin and Alamar blue were from HiMedia Labs, Mumbai, India. Fetal bovine serum of South American origin was from GIBCO, Invitrogen, NY, USA. Power SYBR Green PCR master mix was obtained from Applied Biosystems, California, USA. Antibodies for IL-6, GAPDH, Cyclin D1, PCNA, p21, MMP-2, MMP-9, TIMP-2, RECK, VEGF, VEGFR2, HIF1α, were purchased from Santa Cruz Biotechnology, USA. pJAK-2^tyr1007/1008, JAK-2, pSTAT-3^tyr705, STAT-3 and histone (H2B) antibodies and BrdU, STAT-3^tyr705, total Cyclin D1 and pVEGFR2^tyr1175 ELISA kits were from Cell Signaling Technology, USA. CD-34 antibody was purchased from Novocastra, Germany. Matrigel was from BD Biosciences, USA. All other reagents used were of analytical grade.