Fig. 2: Assembloids confirm the effects of IFN-γ on exfoliating KRT20-traced mature colonocytes and promoting differentiation of progenitors into a new KRT20+ lineage.

a H&E staining (×200 magnification) demonstrating the effect of IFN-γ on crypt formation in assembloids, with quantification on the right. Scale bar: 100 μm. n = 11 from three biological replicates. b KRT20, Ki67, and cleaved-caspase 3 were evaluated via multiplex IF staining (×200 magnification) in assembloids treated with or without IFN-γ. Channel composition on the right. Quantifications are shown in (c–e). Scale bar: 100 µm. n = 4 biological replicates. f Scheme of KRT20/AXIN2 lineage tracing. g, h Confocal microscopy images (×600 magnification) of whole-mount staining of assembloids co-stained with actin/phalloidin derived from g Krt20CreERT2/Rosa26-tdTomato mice or h Axin2CreERT2/Rosa26-tdTomato mice. KRT20 (g) or AXIN2 (h) tracing was induced by 800 nM tamoxifen to label the differentiated lineages (g) or progenitor lineages (h) followed by treatment with or without IFN-γ for 24 h, with quantifications on the right. The box represents the interquartile range (IQR), bounded by the 25th percentile (lower edge) and the 75th percentile (upper edge). The center of the distribution is indicated by the median (solid line within the box). The minimum and maximum values are marked by the whiskers, extending from the lower and upper edges of the box, respectively. n = 5 individual assembloids. Scale bar: 100 µm. i Co-staining of Krt20 and RFP (×600 magnification) was performed in AXIN2-traced untreated or IFN-γ-treated assembloids derived from Axin2CreERT2/Rosa26-tdTomato mice. The percentage of AXIN2-traced KRT20+ cells in all KRT20+ cells was quantified (j). n = 3. Scale bar: 100 µm. k Immunofluorescence staining (×200 magnification) of RFP, C-CASP3, and E-cadherin was performed in assembloids derived from Krt20CreERT2/Rosa26-tdTomato mice. KRT20 tracing was induced by 800 nM tamoxifen to label the differentiated lineage, followed by treatment with or without IFN-γ for 24 h. Scale bar: 100 µm. The experiment was repeated independently three times with similar results. The scheme of KRT20 lineage tracing is presented in the upper panel. All p-values above were determined by Student’s t-test (two-sided). All data are represented as mean ± SD. See also Supplementary Fig. 3. Source data are provided as a Source Data file.