Fig 2: Clusterin expression in neuroendocrine cells.(A) Double immunofluorescence staining of oxyntic mucosa from control rats showing CLU (green) expression in ECL cells (HDC-positive (red)) and A-like cells (ghrelin-positive (red)). A split image of the green (CLU) and red (HDC) fluorescences separately, and a higher magnification of a few dual positive cells (inset), are shown to clearly illustrate the co-expression of CLU in neuroendocrine cells, representative for all species. (B) Double immunofluorescence staining of oxyntic mucosa from control rats showing no CLU (green) expression in parietal cells (H+/K+-ATPase ß (HK)-positive (red)). (C) Double immunofluorescence staining of oxyntic mucosa from wild-type control mice showing CLU (red) expression in ECL cells (HDC-positive (green)). (D) Double immunofluorescence staining of oxyntic mucosa from wild-type control mice showing CLU (red) expression in mucous neck cells (GSII-positive (green)). (E) Double immunofluorescence staining of oxyntic mucosa from control Mongolian gerbils showing CLU (green) expression in ECL cells (VMAT2-positive (red)). (F) ISH and IHC of human oxyntic mucosa showing clusterin (brown and green) expression in scattered single cells partially overlapping with the neuroendocrine marker CgA (red). Inset shows high power view of single CLU-positive cells. Nuclei were counterstained with hematoxylin (blue) or DAPI (blue). The basal zone (~100 µm from the gland bottom) is highlighted with a dotted line. Scale bars = (B, D, F middle column) 100 µm; (A, C, E, F left and right column) 50 µm; (F inset) 20 µm.

Fig 3: TGEV-encoded NSP10 and NSP16 mediate cell proliferation and Lgr5 ISCs fate.a Cell proliferation (KI67), CD24 cells number and Notch signaling factors DII4 and Hes5 were analyzed in TGEV gene stable cell lines. Venn diagram depicts the numbers of decreased cell proliferation and CD24 cells number and down-regulated Notch signaling identified by using FACS and western blot. b FACS for KI67 in TGEV NSPs stable cell lines. c Notch factor and intestinal epithelium markers Muc2, SI, CgA, and CD24 were tested by western blot in TGEV NSPs stable cell lines. d FACS for CD24 in TGEV NSPs stable cell lines.

Fig 4: Goblet cells and enteroendocrine cells in the jejunum of d 0 and 7 piglets. Representative immunohistochemical images of piglet jejunum show goblet cells (A and B), enteroendocrine cells (E and F). Goblet cells are blue both in villus and crypt using AB-PAS staining and are marked by arrows. Enteroendocrine cells are brown in villus and crypt using the anti-CgA antibody and are marked by arrows. Quantitative analysis of goblet cells and enteroendocrine cells was displayed (C and D) and (G and H). The values in the figure are expressed as the mean and SEM (n = 18); differences were assessed by Student's t-test. Values at P < 0.10 show a tendency toward differing (100× magnification; scale bar, 200 µm). AB-PAS = Alcian blue-periodic acid-Schiff staining; CgA = Chromogranin A.

Fig 5: Goblet cells and enteroendocrine cells in jejunal enteroids on d 0 and 7. Representative immunohistochemical images of piglet jejunal enteroids show goblet cells (A and B) and enteroendocrine cells (D and E) (400× magnification; scale bar, 50 µm). Goblet cells are blue using AB-PAS staining and are marked by arrows. Enteroendocrine cells are brown using the anti-CgA antibody and are marked by arrows. Quantitative analysis of the ratio of goblet cells, and enteroendocrine cells was displayed (C and F). Cell nuclei were stained with hematoxylin. The ratio of the number of positive cells to total cell nuclei stained with hematoxylin is calculated. Values in the figure are expressed as means and SEM (n = 4); differences were assessed by Student's t-test and denoted as follows: *P < 0.05. Values at P < 0.10 show a tendency toward differing. CgA = chromogranin A; AB-PAS = Alcian blue-periodic acid-Schiff.