Fig 1: Development of intestinal metaplasia and ectopic gastric gland in aged stCldn18–/– mice. (A) Expression levels of Cldns in the stomach from stCldn18+/+ (n = 6), and stCldn18–/– (n = 7), mice at 100 w.o. quantified by qRT-PCR. The expression levels of Cldn2, 4, 7, and luCldn18 were significantly increased in the stomach of stCldn18–/– mice at old age. Gene expressions were normalized to GAPDH. Results are expressed as mean ± SD. Comparisons between 2 groups were performed using Student’s t test, and differences with P < .05 were considered statistically significant. n.s., not significant. *P < .05. (B) Immunofluorescence micrographs for Cldn2 or Cldn7 (green) co-stained with E-cadherin (red) and with or without villin (blue) in the stomach from stCldn18+/+ and stCldn18–/– mice (around 50 w.o.). Representative images from at least 3 independent experiments are shown. Scale bars = 100 μm. (C) Hematoxylin and eosin–stained images of the ectopic gastric gland in aged stCldn18–/– mice. Ectopic gastric glands were found in 1 of 3 old stCldn18–/– mice by examining stomach tissue slices. Right panels are magnified images of the boxed regions in the left panels. Representative images from at least 2 independent experiments are shown. Scale bars = 100, 50, 10 μm, left to right panel. (D) (Left) Immunofluorescence micrographs for TFF2 (green) and Pepsin C (blue) as SPEM cell markers co-stained with CD44 (red) and DAPI (white) in the ectopic gastric gland from stCldn18–/– mice (around 50 w.o.). (Right) Representative immunofluorescence micrographs for TFF2 (green), and Pepsin C (blue) as SPEM cell markers co-stained with Ki-67 (red) and DAPI (white) in the ectopic gastric gland from stCldn18–/– mice (around 50 w.o.). Right panels are magnified images of the boxed regions in the left panels. Representative images from at least 2 independent experiments are shown. Scale bars = 100, 50 μm, left to right panel. (E) (Left) Immunofluorescence micrographs for Cldn2 (green) co-stained for E-cadherin (red) and villin (blue) in the ectopic gastric gland from stCldn18–/– mice (around 50 w.o.). Right panels are magnified images of the boxed regions in the left panels. Representative images from at least 2 independent experiments are shown. Scale bars = 100, 50 μm, left to right panel. (Middle, Right) Immunofluorescence micrographs for Cldn4 or Cldn7 (green) co-stained for E-cadherin (red) in the ectopic gastric gland from stCldn18–/– mice (around 50 w.o.). Right panels are magnified images of the boxed regions in the left panels. Representative images from at least 2 independent experiments are shown. Scale bars = 100, 50 μm, left to right panel.