Mda mb 231 breast cancer cell line

The human MDA-MB-231 breast cancer cell line (ATCC, Rockville, MD) was maintained in RPMI-1640 medium (HyClone^TM), containing 10% FBS (Gibco) and 1% Penicillin-Streptomycin (Gibco). IL-6 (PeproTech, USA) was used to activate JAK/STAT signaling at working concentrations of 20–60 ng/ml for 6 hours. Cells were treated with the JAK2 inhibitor AG490 (Sigma-Aldrich) and the Akt inhibitor MK-2206 (Selleck, USA) for 24 hours. The Rho inhibitor Rhosin hydrochloride (Tocris Bioscience), Rac1 inhibitor NSC23766 (Santa Cruz Biotechnology) and Cdc42 inhibitor ML141 (Santa Cruz Biotechnology) were reconstituted in DMSO. Small interfering RNA (siRNA) targeting Gramd1b (5′GCUCUUAGAGUCCCAACAATT3′; 3′TTCGAGAAUCUCAGGGUUGUU5′) was designed and synthesized by Singapore Advanced Biologics Pte. Ltd. (SABio, Singapore). si-Gramd1b-2 (Ambion, AM16708) was used to rule out off-target effects of siRNA for Gramd1b. Non-targeting siRNA (Ambion) was used as a negative control. Cells were transfected with siRNA using the transfection reagent Lipofectamine 3000 (Invitrogen, USA) as per the manufacturer’s instructions.

The MDA-MB-231 breast cancer cell line was from the American Type Culture Collection (ATCC, Manassas, VA) and maintained as described [27] (link). The identity of the line was confirmed by The Research Animal Diagnostic Laboratory (RADIL) at the University of Missouri, Columbia, MO (http://www.radil.missouri.edu), using a PCR based method that detects 9 short tandem repeat (STR) loci, followed by comparison of results to the ATCC STR database. A cell bank of defined passage was established and cells were propagated for no more than ten passages in culture.

MDA-MB-231 breast-cancer cell line was obtained from American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM), high-glucose medium purchased from Gibco (Gaithersburg, MD, USA) containing FBS 10% and 1% penicillin/streptomycin. Cells were maintained in a humidified atmosphere of 95% O₂ and 5% CO₂ incubator at 37 °C for normal conditions. For hypoxia conditions, cells were incubated for a specific amount of time in a hypoxia chamber (Stemcell Technologies catalog #27310) with 1% O₂, 5% CO_2, and balanced with N₂, and then treated with DMOG (purchased from Sigma) at a final concentration of 0.5 mM to mimic hypoxia. Cells with low passage numbers were used in all experiments in order to get accurate results.

The human MDA-MB-231 breast cancer cell line was from the American Type Culture Collection (Rockville, MD) and cultured in an L15 medium containing 10% fetal bovine serum (Bioind, Kibbutz Beit-Haemek, Israel). Human umbilical vein endothelial cells (HUVECs) were a kind gift from the General Hospital of Tianjin Medical University. The HUVECs were cultured in VascuLife basal medium supplemented with cytokines. Mouse mammary carcinoma (4T1) cells were obtained from the Chinese Academy of Sciences Cells Bank (Shanghai, China) and cultured in Dulbecco’s modified Eagle’s medium (DMEM) (high glucose) medium with 10% fetal bovine serum. Firefly luciferase-labeled 4T1 cells (4T1-Luc) were produced using Recombinant Lentivirus (GenePharma, Shanghai, China) according to the manufacturer’s instructions. For hypoxic cell culture, cells were grown in an L15 starvation medium containing1% FBS for 12 h before the administration of 1. The cells were then placed into a hypoxic incubator filled with high-purity nitrogen (>99.999%).
Compound 1 was dissolved in dimethyl sulfoxide (DMSO) and, subsequently, a sterile medium for cell treatment. The final concentration of DMSO was 0.1%.

MDA-MB-231 breast cancer cell line was obtained from the American Type Culture Collection. Cells were maintained in Dulbecco’s modified Eagle’s minimal essential medium (DMEM), supplemented with 10% fetal bovine serum and antibiotics (100 U/ml penicillin and 100 U/ml streptomycin) at 37˚C in a humidified atmosphere of 5% CO2. Resveratrol was purchased from Sigma Aldrich (St. Louis, MO, USA), and dissolved at 80 mmol/l concentration, and diluted with DMEM to 100 μM working concentration.

The non-aggressive ERα-positive MCF-7 breast cancer cell line and aggressive triple-negative MDA-MB-231 breast cancer cell line were purchased from the American Type Culture Collection (Manassas, VA, USA). The cells were cultured at 37 °C in an atmosphere of 5% CO₂ and 95% air in Dulbecco′ s Modified Eagle Medium (DMEM), supplemented with 10% fetal bovine serum.