96 well plate luminometer

Commercial scrambled, 14-3-3η siRNA, miR-16 mimic, and anti-miR-16 are listed in Supplementary Table S1. The pcDNA-3.1-14-3-3η-FLAG plasmid that overexpressed both 14-3-3η and FLAG was created by inserting the coding sequences of 14-3-3η (YWHAH, 741 bp) into pcDNA3.1 plasmid, followed by adding a FLAG-tag at its N-terminal (Generay Biotech Co. Ltd., Shanghai, China). Cells were seeded in 6-well plates at a density of 1 × 10⁵ per well, followed by transient transfection using the Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s protocol. After transfection, cells were cultured in fresh medium supplemented with 10% FBS for another 24 h before being used for other experiments. For the luciferase reporter assay, the pGL3-14-3-3η 3′-UTR (wild type, WT; or mutant, MT)-Luc constructs were synthesized by Shuntian Bio Co. (Shanghai, China). The plasmid phRL-tk containing the Renilla luciferase gene was purchased from Promega (Madison, WI, USA). As we described previously^{18 (link),20 (link)}, after cells were plated in 24-well culture dishes for 48 h, they were co-transfected using Luc constructs plus miR-16 mimic. Cells were then lysed with passive lysis buffer, and the lysates were analyzed immediately using a 96-well plate luminometer (Berthold Detection System, Pforzheim, Germany)^{53 (link)}.