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. 2017 Jun 12;8:671. doi: 10.3389/fimmu.2017.00671

Figure 4.

Figure 4

Cell polarization increases the responsiveness to interferon (IFN)-λ. (A) intestinal epithelial cell lines (IECs) harboring Mx2tRFP were cultured on transwell inserts for 3 and 21 days. Cells were treated with 500 U/ml IFN-β or 20 ng/ml IFN-λ3. As indicated, IFN-λ3 stimulation was done together with 750 µM valproic acid (VPA), 500 nM MS275, and 500 nM I-BET151. Mx2tRFP expression was determined 24 h after stimulation by fluorescence microscopy. Representative images are shown. Data are representative of three independent experiments. (B) IEC-Mx2Luc-10 cells containing the Mx2-Luciferase reporter were cultured on transwell inserts for 3 and 21 days as indicated. Cells were treated with 500 U/ml IFN-β or 20 ng/ml IFN-λ3. IFN-λ3 stimulation was done together with 750 µM VPA, 500 nM MS275, and 500 nM I-BET151 as indicated. Luciferase activity was determined 20 h after stimulation (n = 3, mean ± SEM). P values were calculated by one-way ANOVA, followed by Tukey’s Multiple Comparison Test (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001). P values are given for differences among stimulated groups and control group (directly above columns) and between IFN-stimulated groups. (C) IECs cultured on transwell inserts for 21 days were treated with 500 U/ml IFN-β (β) and 20 ng/ml IFN-λ3 (λ) for 16 h. RNA was isolated, and qRT-PCR was used to determine the expression of ISGs. Fold induction after normalization to β-Actin is depicted (n = 3, mean ± SEM). P values were calculated by Mann–Whitney U test (ns, not significant). (D) IECs cultured on standard cultures dishes were treated with increasing concentrations of IFN-β and IFN-λ3 for 1 h. Western blot analysis was performed using antibodies directed against P-Y701 STAT1 and β-Actin. (E) IECs cultured on transwell inserts were treated with 500 U/ml IFN-β and 20 ng/ml IFN-λ3 for 1 h. Cells were lysed, and protein extracts were analyzed by Western blotting using antibodies directed against P-Y701 STAT1 and total STAT1 protein. Results of two independent experiments are shown. (F) IECs were cultured on standard plastic dishes (2D) or on transwell inserts (3D) for 21 days. RNA was isolated, and qRT-PCR was used to determine the expression of IL28R and IFNAR2. Receptor expression was normalized to β-Actin. P values were calculated by Mann–Whitney U test (*P ≤ 0.05).