Reduced organoid growth and progressive loss of PCs after IFN-γ exposure. (A) Organoid number after prolonged culture in presence of indicated concentrations of IFN-γ. Mean of n = 3 independent cultures ± SD is shown, experiment was performed once. All concentrations caused a significantly reduced growth compared with controls (passage 1, P < 0.05; passage 2, P < 0.001, as determined by Student’s t test). (B–D) Time-course analysis of organoids stimulated with 1 ng/ml IFN-γ for the indicated times. (B) Dissociated organoid cells were analyzed by FACS after cleaved Caspase-3 staining. Mean percentage of positive cells from n = 3 independent experiments ± SD is shown; **, P < 10−2; ***, P < 10−4; Student’s t test. See Fig. S1 A for original FACS data. (C) Quantification of epithelial lysozyme content by Western blot. Experiment was performed in n = 3 parallel cultures; mean normalized signals ± SD are shown; *, P < 0.05; **, P < 0.01; Student’s t test. Results were reproduced twice independently. (D) qPCR analysis for marker gene expression of PCs (Lyz1, Defa6), stem cells (Lgr5, Olfm4) and the IFN-γ target gene Irf1. Mean normalized expression values (n = 3 parallel cultures ± SD) are shown. Results are representative for two independent experiments. (E) In situ hybridization analysis of Defa6 expression after IFN-γ treatment (5 ng/ml for 2 d). Overview (top) and magnified images (bottom) are shown that are representative for 3 independent experiments. (F) Immunostaining of membrane markers: CD24 on PCs (red) and the IFN-γ target MHC2 (green) after IFN-γ stimulation (1 d, 1 ng/ml). Note the ubiquitous MHC2 induction also on degranulated PCs (white arrowheads). Images are representative for two independent experiments. (G) Organoids were stimulated for the indicated times (1 ng/ml IFN-γ) before quantification of CD24-positive cells by FACS. Mean cell number (of n = 2 independent experiments ± SD) is shown. *, P < 0.05 as determined by Student’s t test. See Fig. S1 B for the original FACS data. (H) Morphology 1 d after treatment with 5 ng/ml IFN-γ and subsequent culture for 4 d in normal medium. Blue arrowheads show reformation of PC granules. Bars, 50 µm. Experiment was repeated three times.
