DNA Transfection and Luciferase Assay

DNA Transfection and Luciferase Assay

The plasmid NF-kB containing the luciferase coding sequence under the control of NF-kB was kindly supplied by Dr. S. Roman. Cells were seeded at a cell density of 104 cells per well in 96-well plates and were transfected on the following day with plasmid NF-kB (Lipofectamine 2000; Invitrogen SA; Izasa Barcelona, Spain). To normalize for transfection efficiency, plasmid RL-TK containing the Renilla luciferase coding sequence (Promega; Madison, WI) was cotransfected as an internal control. At 24 h posttransfection, the medium was replaced by DMEM with 0.2% FCS. Cells were exposed to TEL, and 1 h later LPS was added (1 pg/mL). After 24 h of incubation, cells were lysed and assayed with a commercial kit (Dual-Luciferase Reporter Assay System; Promega).

DNA Binding Activity of NF-kB

The DNA-binding activity of NF-kB in nuclear extracts was measured (TransAM kit; Active Motif; Carlsbad, CA), according to the instructions of the manufacturer.

Apoptosis Assay

The effect of TEL in the rate of apoptosis in RAW 264.7 and MLE-12 cells was assessed (Cell Death Detection ELISA PLUS System; Roche Diagnostics; Mannheim, Germany). Cells were cultured for 24 h at a density of 5 X 103 cells per well in 96-well plates. Culture media were replaced by DMEM supplemented with 1% FCS, and some cultures received 10 pg/mL TEL 1 h before the induction of apoptosis. RAW 264.7 cells were induced with LPS for 24 h, or with camptothecin (2 pg/mL) [Sigma] for 5 h. MLE-12 cells were induced for 24 h with supernatants from unstimulated and LPS-stimulated RAW 264.7 cells, or with camptothecin. Cells were washed with phosphate-buffered saline (PBS) solution and processed according to the instructions of the manufacturer.

Mouse Model of LPS-induced Lung Injury

Female BALB/c mice, 10 to 12 weeks old, were obtained (Technical Services of the University of Granada; Granada, Spain). The experiments were approved and supervised by the local ethics committee at the University of Granada. Animals were exposed for 20 min to an aerosol of LPS (500 pg/mL) using a custom-built chamber that was directly connected to an air nebulizer (Miko; CA-MI snc; Parma, Italy) that produces particles in the range of 1 to 5 pm at an air flow of 7 L/min.

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