Dual luciferase assay kit

HEK293T cells were transfected with the p55-A2-Luc luciferase reporter plasmid, internal control pTK-Green Renilla plasmid, NOD2 plasmids or control empty plasmids using Lipofectamine 2000^TM (Thermo Fisher Scientific) according to manufacturer’s recommendations. L18-MDP stimulation (200 ng/ml, Invivogen) was performed for 7 h followed by measurement of luciferase activity using the Dual Luciferase Assay Kit (Biotium). To study NF-kB activity in VCP knockdown HCT116 cells, cells were transfected with the p55-A2-Luc luciferase reporter plasmid and pTK-Green Renilla plasmid by lipofectamine 3000^TM (Thermo Fisher Scientific) 24 h after siRNA treatment. L18-MDP stimulation (200 ng/ml, Invivogen) was performed for 4 and 8 h after 72 h of siRNA transfection . To screen NOD2 KO HCT116, the NF-κB luciferase reporter assay was performed as described for HEK293T cells.

Luciferase assays were conducted as described previously [22 (link)]. Briefly, approximately 2.5 x 10⁴ cells were seeded per well in a 24-well plate. Transfections were conducted using Lipofectamine 2000 following manufacturer’s guidelines. Each reaction contained 50 ng of the luciferase reporter plasmid, and 2 ng pLRL-SV40 Renilla, which served as a transfection control. Where indicated, 50 ng of pcDNA3.1-β-catenin S45F [28 (link)], 50 ng of pME18 Lef [28 (link)], and 50 ng of pLV hU6-sgRNA hUbC-dCas9-KRAB-T2a-Puro were added to the transfection. The phU6-sgRNA plasmids encoding the guide RNAs (25 ng each) were included as indicated. Total concentration of DNA was adjusted to 2 μg per reaction using pBluescript (Stratagene). Transfection mixtures were incubated on cells for 6 h, after which the media was replaced with normal growth media. Each reaction was conducted in quadruplicate. After 24 h, cells were lysed in 200 μl passive lysis buffer and luciferase levels were measured using the dual luciferase assay kit (30005–2; Biotium) on a Glomax 20/20 single chamber luminometer (Promega).

Luciferase assays were performed using a dual-luciferase assay kit (Biotium, Fremont, CA, USA) and readouts were collected using the Molecular Devices iD3 plate reader (San Jose, CA, USA). Cells were co-transfected with a STAT3-responsive luciferase reporter (pGAS-luc), and either vector or TrkA (pCMV5-TrkA) plasmid for 28 h before serum starvation for 16 h. Then, cells were stimulated with 100 ng/mL β-NGF for 4 h before the addition of lysis buffer. Lysates underwent mechanical lysis by scraping followed by trituration with a pipette to ensure complete lysis. Firefly luciferase activity was measured as previously described [56 (link),63 (link)]. Results are represented as mean ± SD. Student’s t-tests were performed using GraphPad Prism 8.