Luc2p nf κb re hygro

For the luciferase assay with NF-κB, pGL4.32 (luc2P/NF-κB-RE/Hygro) vector containing five copies of NF-κB response element (Promega) was used. The hDPCs were transfected with the reporter vector and miR-146b mimic or miR-146b NC using Lipofectamine 3000 transfection reagent (Thermo Fisher Scientific) and then stimulated with LPS (100 ng/mL). For the luciferase assay with RELA 3′-UTR reporter vector, synthesized RELA 3′-UTR containing wild-type or mutated hsa-miR-146b-5p target sequences (400 bps each; Eurofins Genomics, Ebersberg, Germany) were inserted into XhoI and HindIII sites of a pMIR-REPORT vector (Thermo Fisher Scientific). The reporter vectors along with miR-146b mimic or miR-146b NC were transfected into hDPCs via Lipofectamine 3000 transfection reagent (Thermo Fisher Scientific). Transfected cells were lysed with a luciferase cell culture lysis reagent and the luciferase activity was measured using luciferase assay system (Promega) and a luminometer (Luminescence PSN; ATTO).

Cells were transfected with the luciferase reporter plasmid pGL4.32[luc2P/NF-κB-RE/Hygro] (Promega, Madison, WI, USA), and stably expressing cells were selected with hygromycin. Before the luciferase assay, cells were plated on 96-well plates at a density of 1.5 × 10⁴ cells/well and cultured in DMEM for 24 h. The cells were then treated with 20 ng/mL of TNF-α (Wako, Osaka, Japan) for 5 h. Luciferase activity was measured by using the Nano-Glo Dual-Luciferase Reporter Assay System (Promega). Each experiment was independently conducted three times, with five replicates for each sample.

HEK293 cells were transfected with the luciferase reporter plasmid pGL4.32[luc2P/NF-κB-RE/Hygro] (Promega, Madison, WI), and stably expressing cells were selected by hygromycin. Twenty-four hours before transfection, cells were plated on 96-well plates (1.5 × 10⁴ cells/well). Transfection with plasmids was performed using FuGENE® HD Transfection Reagent (Promega). Twenty-four hours after transfection, cells were treated with tumor necrosis factor-α (TNF-α, 20 ng/ml) (Wako, Osaka, Japan) for 5 h. Then, luciferase activity was measured by using a Nano-Glo® Dual-Luciferase® Reporter Assay System (Promega). Each experiment was independently performed three times with five replicates of each sample.