AhR-dependent and -independent pathways to MK maturation identified using luciferase AhR reporter gene assay. (A) WNT and FLT3 signaling was not relevant to the enhancement of imMKCL maturation by Wnt-C59 and TCS 359. Shown are the effects of inhibitors of WNT and FLT3 signaling on Venus fluorescence intensity in β1-tubulin–Venus reporter imMKCLs. (B) Construction of a luciferase AhR reporter gene assay. The human CYP1A1 promoter region containing 2 AhR-recognition sequences was inserted into the XhoI-BglII site of pGL4.27. Huh7 cells were cotransfected with hCYP1A1-pGL4.27 and pRL-TK-pGL4.74 vectors overnight using Lipofectamine 3000, after which they were incubated with compounds for 24 hours, followed by treatment with 1 nM 2,3,7,8-tetrachlorodibenzo-p-dioxin overnight. Firefly and Renilla luciferase activity was evaluated using a Wallac Arvo Sx 1420 (PerkinElmer) with a Dual-Glo Luciferase Assay kit. (C) Dose-response curves of the candidate compounds obtained with the luciferase AhR reporter gene assay. The candidate compounds were categorized into 2 groups based on whether their dose-response curves were sigmoid or nonsigmoid.