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Figure 5. AhR-mediated Ccno expression is prevented by Notch signalling and requires normoxic air exposure.(a) Quantification of acetylated α-tubulin fluorescence intensity from entire wells of AhR-sufficient mTEC cultures grown for 8 days in ALI, submersion or ALI under hypoxic condition. Fluorescence intensity of ALI condition was set as 100%. Mean±s.e.m.; Student's t-test (unpaired, two-tailed). n=3 wells per group, data from three independent experiments. (b) mRNA expression levels of Ccno in Ahr+/+ mTEC cultures grown in ALI, submersion or ALI under hypoxic condition at indicated days of treatment. Values are normalized to Hprt1. Mean±s.e.m.; two-way analysis of variance (variables: treatment and time). Data representative of three independent experiments. (c) Chromatin immunoprecipitation (ChIP) analysis of AhR interaction with the Ccno promoter in Ahr+/+ mTEC cultures grown in ALI, submersion or ALI under hypoxic condition at indicated days of treatment. Mean±s.e.m.; Student's t-test (unpaired, two-tailed). Data representative of three independent experiments. (d) Quantification of acetylated α-tubulin fluorescence intensity from entire wells of AhR-sufficient and AhR-deficient mTEC cultures at indicated days of ALI in presence or absence of DAPT. Fluorescence intensity of Ahr+/+ mTEC culture at 3 days of ALI was set as 100%. Mean±s.e.m.; Student's t-test (unpaired, two-tailed). Data representative of three independent experiments. (e) mRNA expression levels of Ccno in Ahr+/+ and Ahr−/− mTEC cultures at indicated days of ALI in presence of DAPT or vehicle control. Values are normalized to Hprt1. Mean±s.e.m.; Student's t-test (unpaired, two-tailed). Data representative of three independent experiments. (f) ChIP analysis of AhR interaction with the Ccno promoter in AhR-sufficient mTEC cultures grown in ALI or submersion in the presence of DAPT or vehicle control. Mean±s.e.m.; Student's t-test (unpaired, two-tailed). Data representative of two independent experiments. Ctrl, control. Image published in: Villa M et al. (2016) Copyright © 2016, The Author(s). This image is reproduced with permission of the journal and the copyright holder. This is an open-access article distributed under the terms of the Creative Commons Attribution license Larger Image Printer Friendly View

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