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. 2018 Apr 17;8:6103. doi: 10.1038/s41598-018-24213-5

Figure 4.

Figure 4

AhR activation is required for 5-HT to induce CYP1A1 mRNA. Caco-2 cells were plated at low density and allowed to differentiate for 10–14 d in medium containing 20% serum before treatments were performed without transfection (a,d,e). When transfection was performed, Caco-2 cells were treated beginning 24 h post-transfection (b,c). (a) Caco-2 cells were treated with vehicle (Con) or AhR antagonists 10 μM CH-223191 (CH) and 10 μM 3′,4′-dimethoxyflavone (3′,4′-DMF) for 1 h before co-treatment of each antagonist with 5-HT (10 μM) for 24 h in serum-free medium. Data represent the relative expression of CYP1A1 mRNA quantified by qPCR. Data were analyzed by 1-way ANOVA followed by Tukey’s multiple comparison’s test (n = 4). (b,c) Caco-2 cells were transfected with control siRNA (si-Con), AhR siRNA (si-AhR), or ARNT siRNA before treatment with 5-HT (10 μM) for 8 h (si-AhR) or 24 h (si-ARNT) in serum-free medium. AhR, ARNT, and CYP1A1 mRNA expression were determined in untreated cells by qPCR and results are expressed as fold change compared to control siRNA. Data analyzed by paired 2-tailed Student’s t-test. Unpaired 2-tailed Student’s t-test was used to compare CYP1A1 mRNA induction after 5-HT treatment in control siRNA vs. AhR or ARNT siRNA transfected cells (n = 3). (d) Caco-2 cells were treated with 5-HT (10 μM) for 4 h in serum-free medium before nuclear and cytoplasmic extracts were isolated and Western blotting was performed for AhR. Nuclear AhR was normalized to Lamin B1 and cytoplasmic AhR was normalized to GAPDH. The ratio between nuclear and cytoplasmic AhR as determined by densitometry was analyzed by paired 2-tailed Student’s t-test (n = 4). Full-length blots are shown in Supplementary Figure S1. (e) Caco-2 cells were treated with 5-HT (10 μM) for 24 h in serum-free medium. Data represent the relative expression of AhR mRNA quantified by qPCR (n = 3). *P < 0.05, **P < 0.01, ****P < 0.0001 vs. untreated, control siRNA, or no 5-HT. ####P < 0.0001 vs. 5-HT treatment without AhR antagonist. n.s. = not significant. Red circle = untreated, blue square = 5-HT.