Prl cmv renilla luciferase plasmid

TOP-flash - luciferase reporter plasmid containing multiple copies of an optimal TCF-binding site was purchased together with FOP-flash (mutated plasmid) from Addgene (Cambridge, MA, USA). pRL-CMV - renilla luciferase plasmid was obtained from Promega (Madison, WI, USA). For knockodown experiments pLKO.1.sh.beta-catenin.2279, a vector containing shRNA sequence against β-catenin (Addgene plasmid # 19762), and pLKO.1 puro, an empty control vector for RNAi approach (Addgene plasmid # 8453), were used.

To generate LGALS1 promoter‐enhancer reporter constructs,11, 12, 13 three fragments spanning nucleotides −500 bp to +67 bp from the LGALS1 transcription start site (promoter region), +450 bp to +1750 bp (enhancer region), and AP‐1 site (TGACTCA)‐mutated enhancer region were synthesized and sequenced by Integrated DNA Technologies, and subcloned into the pGL4 promoterless reporter vector (Promega), generating pGal, pGal + AP‐1 and pGalΔAP‐1. The pRL‐CMV Renilla luciferase plasmid (Promega) was used as internal control. The Dual‐Luciferase Reporter Assays System (Promega) was used to measure the activity of firefly and Renilla luciferase. Cells were transfected with plasmid DNA using Lipofectamine LTX with Plus Reagent (Thermo Fisher Scientific) following the manufacturer's protocols.
The Pathway Profiling SEAP System (TAKARA BIO) was utilized to detect pathways in response to corticosteroids following the manufacturer's recommendations. After transfection, the medium was collected, and secreted alkaline phosphatase was measured using the chemiluminescent alkaline phosphatase detection kit Great EscAPe™ SEAP (TAKARA BIO) according to the manufacturer's procedures.

SNU-449 and HLE Cells were plated in triplicate in 24-well plate at 70–80% confluency and were co-transfected with 500ng of TEAD luciferase reporter plasmid (8xGTIIC-luciferase, Addgene plasmid #34615), 20ng of pRL-CMV Renilla luciferase plasmid (Promega, Madison, WI) and 1μL Lipofectamine 2000 per well (Invitrogen). The culture medium was replaced by Opti-MEM^® Reduced Serum. After 4-hour incubation, the transfection mixture was replaced by culture medium with 0.1%DMSO, 10μM XAV-939, or 10μM G007-LK. At 48 hours, the cells were rinsed in 1X PBS and harvested with Passive Lysis Buffer in a new tube on ice. Luciferase activity was measured using the Dual-Luciferase^® Reporter Assay System (Promega), according to the manufacturer’s protocol.

Dual-luciferase reporter assays were performed in HEK293T cells to detect the effects of oyster IKKα/β-2 protein on transcription from the NF-κB, TNFα, ISRE, and IFNβ promoters using Myc-fused protein expressing vectors. Briefly, cells in 24-well plates (Corning, USA) were transfected with 0.1 μg of reporter gene plasmids, 0.01 μg of pRL-CMV Renilla luciferase plasmid (Promega), and varying amounts of expression plasmids or empty expression vector (as a control). The pRL-CMV Renilla luciferase plasmid was used as an internal control. At 24–48 h post-transfection, the Dual-Luciferase Reporter Assays System (Promega) was used to measure the activities of firefly and Renilla luciferases according to the manufacturer's instructions. Experiments were performed in triplicate.

Human LGALS1 promoter (−500 bp to +67 bp from the LGALS1 transcription start site)11, 16, 17 was synthesized and sequenced by Integrated DNA Technologies and subcloned into the pGL4 vector (Promega). Luciferase reporter construct containing 3 consensus hypoxia‐responsive elements (HREs) was obtained from Addgene. The pRL‐CMV Renilla luciferase plasmid (Promega) was used as an internal control. The dual‐luciferase reporter assays system (Promega) was used to measure the activity of firefly and Renilla luciferase. Cells were transfected with plasmid DNA was transfected using Lipofectamine LTX with Plus Reagent (Thermo Fisher Scientific) following the manufacturer's protocols.