Pnf κb seap

Jurkat cells (1 × 10⁵) were electroporated with 5 μg of pNFAT-SEAP or pNF-κB-SEAP (Takara Bio, Shiga, Japan) using a GenePulser Xcell apparatus (Bio-Rad, Tokyo, Japan) by a preinstalled procedure. The cells with pNFAT-SEAP or pNF-κB-SEAP were treated with 1 μM FK506 or 20 μM RCAN-11R/scRCAN-11R for 1 h and with 200 nM PMA and 4 μM ionomycin for an additional 12 h. The supernatant was transferred into a new tube and mixed with a Great EscAPe Chemiluminescence Detection Kit (Takara Bio) in cuvettes for luminometry.

The ARPE-19 cells (3 × 10⁴/well) were plated and maintained in the DMEM/F-12 medium with 10% FBS in 24-well dishes for 24 h. To measure the NF-κB activity, the ARPE-19 cells were cotransfected with pCMV-luciferase (Promega, Milan, Italy) and either pTAL-secreted alkaline phosphatase (SEAP) or pNF-κB-SEAP (Clontech, San Jose, CA, USA) at a ratio of 1 : 4 for 4 h using the polycationic detergent Lipofectamine Plus (Invitrogen-Gibco) according to the manufacturer's instructions. The ARPE-19 cells were maintained for 20 h and subsequently preincubated with or without 100 μM PUGNAc for 1 h. The cells were further incubated with or without 50 μM silibinin for 24 h before stimulation with TNF-α (20 ng/mL) for 24 h at 37°C. For each treatment, the experiments were performed in triplicate. The SEAP activity was determined in the culture supernatants, and the luciferase activity was measured in the cell lysates to normalize the transfection efficiency. The luciferase activity was assessed with the Promega Dual-Luciferase Reporter 1000 Assay System.

To monitor the activation of NF-κB and AP-1 signal transduction pathways, the plasmids (pNFκB-SEAP or pAP1-SEAP) were purchased from Clontech and were transiently transfected into MARC-145, MARC-2a, or MARC-N cells by the use of Lipofectamine LTX (Invitrogen). Briefly, 4 μg of each plasmid DNA, 12 μL Lipofectamine LTX, and 4 μL Plus reagent were used to transfect cells, seeded in a well of 6-well plate. Since these transfected plasmids contain the secreted alkaline phosphatase (SEAP) gene as a reporter, culture supernatants were collected 72 h after transfection, and SEAP activity was detected in by the Great Escape SEAP Chemiluminescence Assay kit (Clontech) using a GloMax 20/20 Luminometer (Promega). The chemiluminescence emitted by a SEAP-activated substrate (CSPD) was measured in relative luminescence units.