Figure 2.

“Memory” effect in IFN-stimulated cells. (A) Intestinal epithelial cell lines (IECs) harboring Mx2tRFP were stimulated with either 10 U/ml interferon (IFN)-β or 25 ng/ml IFN-λ3 for 20 h. Cells were infected with vesicular stomatitis virus-GFP (MOI 0.02) for 1 h and imaged by confocal microscopy 20 h post-infection. Representative pictures are shown. Data are representative of three independent experiments. (B) IECs were stimulated for 24 h with 25 U/ml IFN-β, and Mx2tRFP-positive and -negative populations were separated by cell sorting. Cells were cultured for 48 h in the absence of IFN, and responder and non-responder populations were restimulated for 20 h with the indicated concentrations of IFN-β and IFN-λ3. Mx2-driven tRFP expression was measured by flow cytometry. Representative FACS dot plots are shown. (C) Frequencies of Mx2tRFP expression in responder and non-responder populations upon IFN restimulation were calculated from two independent experiments (n = 4, mean ± SEM). P values were calculated by Mann–Whitney U test (*P ≤ 0.05). (D) Sorted Mx2tRFP-negative and Mx2tRFP-positive cell populations were cultured for 48 h in the absence of IFN. Both populations were stimulated for 1 h with 50 U/ml IFN-β, fixed, and processed by immunofluorescence staining against P-Y701 STAT1. Control column represents Mx2tRFP-positive cells without IFN treatment. Images were obtained by confocal microscopy, and the percentage of cells showing activated STAT1 in the nucleus was calculated (n indicates the number of analyzed cells). Representative pictures are shown. DAPI staining indicates localization of cell nuclei. Dot plots show representative Mx2tRFP expression analysis from (B) after restimulation with 50 U/ml IFN-β for 20 h.