Fig. 2: Changes in expression levels of differentiated intestinal epithelial cell-associated markers in mIOs after treatment with Amuc_1409.

a His-tagged Amuc_1409 was cloned into the pET30a(+) vector using restriction endonuclease sites (NdeI/XhoI) (left panel) and the purified His-tagged Amuc_1409 protein (Amuc_1409^*) was stained with Coomassie brilliant blue on a 10% SDS-PAGE gel under reducing conditions (right panel). Lane 1 shows the molecular size of standards with their apparent molecular weights (marker) in kDa. Lane 2 shows Amuc_1409^*. b Schematic diagram of Amuc_1409^* treatment (Tx.) schedule in young mIOs. mIOs from SI crypt of young (3-month-old) mice were treated with Amuc_1409^* (8 nM) on day 0 and day 2 after the second subculturing passage and harvested on day 4. qRT-PCR results showing the relative mRNA expression of secretory progenitor cell markers (c), enteroendocrine cell markers (d), Paneth cell markers (e), goblet cell markers (f), mucin markers (g), and mature enterocyte markers (h) in young mIOs treated with or without Amuc_1409^* (8 nM). Representative IF staining (left panel) for the enteroendocrine cell marker (ChgA) (i), Paneth cell marker (Lysozyme) (j), and goblet cell marker (Muc2) (k) in young mIOs treated with or without Amuc_1409^* (8 nM). ChgA, Lysozyme, and Muc2 (green); DAPI (nuclei, blue). White scale bar, 50 μm. Yellow scale bar, 20 μm. Quantification data of positive cells for each marker per organoid are presented (i–k, right panel). All data are presented as the mean ± SEM. In (c–k), a different symbol indicates a data point representing each biological replicate from independently established organoid lines derived from distinct mouse litters (n = 3 biologically independent mice). In (c–h), each biological replicate includes two or three technical replicates. In (i–k), positive cells for each marker were counted in 20 organoids per group from each biological replicate. Statistical analyses were performed using two-tailed Student’s t test (c–k) (*p < 0.05, **p < 0.01, and ***p < 0.001 vs control group). Source data, including the exact p values, are provided as a Source Data file.