Multi-Level Interactions between the Nuclear Receptor TRα1 and the WNT Effectors β-Catenin/Tcf4 in the Intestinal Epithelium
Figure 2
The Wnt3a ligand is not sufficient to impair TRα1 transcriptional activity ex vivo.
The primary cultures of intestinal epithelial cells were treated with 10 ng/ml of Wnt3a and/or 10−7 M of T3 for 24 hours. (A) The number of proliferating cells in the different experimental conditions was analyzed by Ki67 immunolabeling; all of the nuclei were labeled by Hoechst. The percentage of Ki67-positive nuclei was determined by counting under a fluorescence microscope (Zeiss Axioplan). The histograms represent the summary (mean ± sd) of the scoring of specific immunolabeling in two independent experiments each conducted in triplicate (n = 50). (B, C) Analysis of β-catenin in intestinal epithelial primary cultures by immunolabelling (B) and WB (C). Cells were in control, T3, Wnt3a and T3+Wnt3a conditions as indicated. Pictures in B show the fluorescent staining of the nuclei (blue), β-catenin (red) and the merging of each simple labeling. Bar: 15 µm. For the WB (C), we used a specific antibody allowing the detection of activated non-phosphorylated β-catenin [54], [55]. Actin was used as loading control. The image is representative of two independent experiments. Each lane represents whole protein extracts (50 µg/lane). (D–F) RT-qPCR analysis to evaluate mRNA levels of Ccnd1, Ctnnb1 and Sfrp2. Results are from three independent experiments, each conducted in duplicate. Values represent fold change ± sd after normalization to the control condition (Ctrl). *: P<0.05, **: P<0.01, ***: P<0.001 by two-tailed Student's t-test (n = 6).