Human il 1β

Two different human lung carcinoma cell lines, LU65 and LU99, and human breast adenocarcinoma cell line MCF7, were obtained from the RIKEN Cell Bank. HUVEC, human lung carcinoma cell line H292, and human ductal breast epithelial tumor cell line T47D were from American Type Culture Collection (ATCC). HUVEC was maintained in endothelial cell culture media (Life Technologies) with endothelial cell growth supplement (Millipore). LU65 and LU99 cells were cultured in DMEM/F-12 + 10% FBS. MCF7, T47D, and H292 cells were in RPMI 1640 + 10% FBS. CHO cells transfected with the control vector or human LOX-1 tetracycline-inducible expression vector were cultured in F12 (Wako Pure Chemical). CHO cells were treated with serum-free medium containing 1 mg/L doxycycline for 16 h prior to the experiments. Cells were stimulated with 100 nM dexamethasone (Sigma-Aldrich, D4902), 10 ng/ml human IL-1β (Miltenyi Biotec, Auburn, CA USA), and 10 ng/ml IL-6 (Miltenyi Biotec), or 1 μg/ml LPS (Sigma-Aldrich, L2880) for 6 h for qPCR analyses or for 3 days for protein analysis.

The monocyte isolation kit (Ord. no 130-091-153; Miltenyi Biotech, Bergisch Gladbach, Germany) was used for negatively selecting the CD14⁺ monocytes from all subjects. Monocytes were cultured for 24 h in RPMI 1640 medium + L-glutamine (Gibco^® Thermo Fisher Scientific) supplemented with 10% heat-inactivated fetal bovine serum (Gibco^® Thermo Fisher Scientific) and 100 IU/ml penicillin/streptomycin (Sigma-Aldrich, Taufkirchen, Germany). Monocytes were treated for 24 h, either with 500 IU/ml human IL1β (Ord. no 130-093-897; Miltenyi Biotech) as an inflammatory stimulant, and/or 1 × 10⁻⁸ mol/L 1,25(OH)₂D₃ (Enzo, Lörrach, Germany) for demonstrating the vitamin D-regulating effect in stimulated monocytes, or 1 × 10⁻⁸ mol/L 1,25(OH)₂D₃ was added alone without IL1β, in comparison to untreated cells, for demonstrating the vitamin D-regulating effect in resting monocytes. Dose and exposure time of 1,25(OH)₂D₃ and IL1β was based upon our previous work (24 (link)).