Pgl4.32 luc2p nf κb re hygro luciferase reporter vector

The pGL4.32 [luc2P/NF-κB-RE/Hygro] luciferase reporter vector (Promega, Madison, WI) was electroporated into RAW264.7 cells by using a Lonza Nucleofector II instrument (Amaxa Biosystems, Germany) as described previously [18 (link)]. Cells were incubated with pinocembrin (0–150 µM) for 2 h and followed by an additional treatment with LPS (1 μg/ml) for 22 h. Cells were washed with PBS and then lysed in 1× passive lysis buffer (100 µl). The luciferase activity from cell lysates was detected using a luciferase assay system (Promega, Madison, WI). Results were expressed as fold induction of control cells.

2×10⁶ LS174T cells in 100 μl transfection buffer (Cell Line Nucleofector Kit V) were co-electroporated with 1 μg pGL4.32[luc2P/NF-κB-RE/Hygro] luciferase reporter vector (Promega, Madison, WI), 0.5 μg expression vector (pSG5-hPXR or pSG5 control), and 0.1 μg pRL-TK vector using Lonza Nucleofector II instrument (program Q-009). For detailed plasmids information, please refer to our previous report (Dou et al., 2012a (link)). The cells were transferred to 48-well plate following transfection and were treated with TNF-α (20 ng/ml, Sigma-Aldrich, St. Louis, MO) alone or in combination with alpinetin (25 μM) for 24 h. The cells were harvested in passive lysis buffer (Promega) and luciferase activity was detected using the dual-luciferase reporter assay system (Promega). Luminescence was detected by Turner Bio-systems Luminometer 20/20n (Turner Biosystems, CA). The results were expressed as the fold induction of the control cells.

The pGL4.32[luc2P/NF-κB-RE/Hygro] luciferase reporter vector (Promega, Madison, WI) was electroporated into LS174T cells or HT-29 cells. The pGL4.32 reporter is a NF-κB reporter vector including NF-κB response elements and firefly luciferase gene. Cells were incubated with ALA (0–25 µM) for 2 h followed by an additional co-incubation with lipopolysaccharide (LPS, 1 µg/ml) for 24 h. The luciferase activity was detected using a luciferase assay system (Promega, Madison, WI).