Journal: Nature Communications

Article Title: Aryl hydrocarbon receptor utilises cellular zinc signals to maintain the gut epithelial barrier

doi: 10.1038/s41467-023-41168-y

Figure Lengend Snippet: a Caco-2 cells were grown to an epithelium in a Transwell® system. At the start of the experiment, the medium in the apical compartment was replaced with MEM containing FITC-dextran 4000 (Merck) together with 3% DSS, 0 or 8 µM zinc, and 0 or 100 nM FICZ as indicated in the figure. Permeability was measured by sampling the medium in the basal compartment and measurement of FITC fluorescence over 24 h ( n = 3 independent experiments). b human ileum organoids were challenged with 60 µM EDTA in presence of FITC-dextran in a medium containing 60 µM zinc and/or 100 nM FICZ. Leakage into the lumen was assessed after 24 h through appearance of FITC fluorescence. c Expression of mRNA for tight junction proteins as measured by qPCR in Caco-2 cells treated with zinc/FICZ/TPA for 24 h as indicated in the figure ( n = 3 experiments). d Expression of mRNA for tight junction proteins and MUC2 as measured by qPCR in human ileum organoids treated with zinc and/or FICZ for 24 h as indicated in the figure ( n = 3 experiments). e Abundance of tight junction proteins, as measured by western blot, in Caco-2 cells treated with Zinc/FICZ/TPA for 24 h as indicated in the figure ( n = 3 experiments). f Immunocytochemistry (left) showing MUC2 ( n = 4 experiments) and OCLN ( n = 4 for control group and n = 3 for the other groups) protein levels in human ileum organoids treated with 0 or 100 nM FICZ and/or 0 or 25 µM zinc for 24 h, as indicated in the figure. Scale bar, 20 µm. Fluorescence levels in all regions containing DAPI and 488 nm staining were quantified using NIS Elements (Version 5.41, Nikon Instruments) and numeric data are shown (right). Statistical analysis of the data was performed using 2-way ANOVA followed by Tukey’s multiple comparison tests ( a ), or 1-way ANOVA followed by Tukey’s multiple comparison tests ( c – f ). Data are presented as mean values and error bars show SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns not significant.

Article Snippet: Human epithelial colorectal adherent Caco-2 cells, a gift from Prof. Paul Sharp, King’s College London, were maintained in Minimum Essential Medium Eagle (MEM, Merck), supplemented with 10% fetal bovine serum (FBS, Merck), 1% 100× MEM Non-essential amino acid solution (Merck), 1% 250 μg/mL Amphotericin B (Merck), and 1% 100 units/mL penicillin (Merck), at 37 °C in a humidified atmosphere of 95% air and 5% CO 2 .

Techniques: Permeability, Sampling, Fluorescence, Expressing, Western Blot, Immunocytochemistry, Staining, Comparison