Cell experiment:

Subconfluent cells (25, 000 cells/well) are seeded in 96-well plates. Cells are treated with ITE at 5, 10 and 20 μM or DMSO (0.1% v/v) in ECM for 2, 4 or 6 days with a change of ECM containing DMSO or ITE every other day (5 wells/treatment). At the end of treatment, cells are incubated with MTT reagent for 4 hr, and solubilized in crystal dissolving solution (100 μL/well) for 20 min. The absorbance is determined at 570 nm using the microplate reader[3].

Animal experiment:

Mice[2]Eight- to 12-week-old female B10.A mice is used in the assay. Daily treatment starts on day 0 and consists of 200 μg of ITE suspended in 0.2 mL PBS, given intraperitoneally. Control mice are similarly treated with 0.2 mL of the vehicle, PBS containing 3.6% DMSO[2].

2-(1′H-indole-3′-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE) is a nontoxic immunosuppressive endogenous aryl hydrocarbon receptor (AhR) ligand [1] [2]. Its EC50 identified via yeast AhR assay is 7.8×10−10 mol/l [3].
AhR is a transcription factor mediating toxic effects of environmental pollutants such as 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD), it is activated by ligand [2].
ITE inhibited TGFβ1-induced myofibroblast differentiation in TGFβ1-challenged cultures of primary human fibroblasts from several distinct tissue types. In primary human orbital fibroblasts, ITE inhibited TGFβ1-induced nuclear translocation of Smad2/3/4 and its subsequent binding to SBE, but it did not inhibit TGFβ1-induced phosphorylation of Smad2/3, Erk1/2, or Akt [4], it inhibited TGFβ1 (1 ng/mL)-induced α-smooth muscle actin (α-SMA) expression, extracellular matrix production [5]. The inhibition of ITE to TGFβ1-induced myofibroblast differentiation in primary human fibroblasts is AhR independent [5].
In Sprague-Dawley rats maintained under a 12 h light/12 h dark cycle and given free access to a solid diet and distilled water, ITE at 1.6 and 8.0 mg/kg bw, significantly increased the CYP1A1 mRNA levels by 5.59 and 68.55 fold, respectively. It indicated that ITE activated AhR activation in placentas. 8.0 mg/kg bw ITE elevated the level of HIF-1a mRNA, induced the levels of VEGF-A, VEGF-B and PIGF mRNA in a dose-dependent manner [6].
References:
[1]. Lindsey F. Nugent, Guangpu Shi, Barbara P. Vistica, et al. ITE, A Novel Endogenous Nontoxic Aryl Hydrocarbon Receptor Ligand, Efficiently Suppresses EAU and T-Cell–Mediated Immunity. Invest Ophthalmol Vis Sci., 2013, 54(12):7463-7469.
[2]. Jaishree Bankoti, Ben Rase, Tom Simones, et al. Functional and phenotypic effects of AhR activation in inflammatory dendritic cells. Toxicol Appl Pharmacol., 2010, 246(1-2):18-28.
[3]. S. Medjakovic and A. Jungbauer. Red clover isoflavones biochanin A and formononetin are potent ligands of the human aryl hydrocarbon receptor. Journal of Steroid Biochemistry & Molecular Biology, 2008, 108:171-177.
[4]. Geniece M. Lehmann, Xia Xi, Ajit A. Kulkarni, et al. The Aryl Hydrocarbon Receptor Ligand ITE Inhibits TGFβ1-Induced Human Myofibroblast Differentiation. American Journal of Pathology, 2011, 178(4):1556–1567.
[5]. Geniece M. Lehmann, Xia Xi, Ajit A. Kulkarni, et al. The Aryl Hydrocarbon Receptor Ligand ITE Inhibits TGF1-Induced Human Myofibroblast Differentiation. The American Journal of Pathology, 2011, 178(4):1556-1567.
[6]. Yanming Wu, Xiao Chen, Qian Zhou, et al. ITE and TCDD Differentially Regulate the Vascular Remodeling of Rat Placenta via the Activation of AhR. PLoS ONE, 2014, 9(1): e86549.