Dual glo stop and glow luciferase assay system

24 h prior to transfection, HeLa cells were seeded at a density of 4 × 10⁴cells per well. 800 ng/well of S100A9-unspliced or S100A9-spliced luciferase reporter gene plasmid and 200 ng/well of pSV40-Rluc as internal standard were transfected using Lipofectamine2000^® (Invitrogen) according to manufacturer′s instructions. For co-transfection with siRNAs, 200 ng/well of reporter gene construct, 200 ng/well of pSV40-Rluc and 20 pmol/well siRNA were used for transfection with Lipofectamine2000. After 24 h, reporter gene activity was determined with the Dual-Glo™ Stop and Glow Luciferase Assay system following the manufacturer’s protocol (Promega) and measured with a Tecan infinite^® M200 reader. Renilla luciferase activity was used to normalize the luciferase activity to the transfection efficacy.

Twenty-four hours prior to transfection, 4×10⁴ HeLa cells per well were seeded in 24-well plates; 400 ng/well of TLR2 or NOX2 constructs and 5 nmol miR-328 mimics and control mimics were used for transfection with Lipofectamine 2000^® (Invitrogen) according to the manufacturer’s instructions. After 24 h, cells were assayed for luciferase activity using the Dual-Glo^™ Stop and Glow Luciferase Assay System (Promega) with a TECAN infinite M200 reader. Renilla luciferase activity was used to normalize the luciferase activity to the transfection efficacy.