Fig 2: Activation of inflammatory signals including CCL28 and CXCL5 in aged stCldn18–/– mice. (A) Expression levels of IL-1ß, TNF-a, and CXCL1 in the stomach from stCldn18+/+ (n = 8, 4, 11, 10) and stCldn18–/– mice (n = 14, 4, 13, 16) (8, 40, 60, and 100 w.o.) quantified by qRT-PCR. Gene expressions were normalized to glyceraldehyde-3-phosphate dehydrogenase (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. *P < .05; **P < .01. (B) Immunofluorescence micrographs for CXCL5 (green) co-stained with a-catenin (red) and DAPI (blue) in the stomach from stCldn18+/+ mice and stCldn18–/– mice (8, 40, 60, and 100 w.o.). CXCL5 signals were clearly observed in the stomach of stCldn18–/– mice after middle-age (40, 60, and 100 w.o.) (arrows). High-magnification images are shown in insets. Representative images from at least 3 independent experiments are shown. Scale bars = low magnification: 50 µm, high magnification: 10 µm. (Right) Expression level of CXCL5 in the stomach from stCldn18+/+ (n = 8, 4, 12, 10) and stCldn18–/– mice (n = 6, 4, 13 16) (8, 40, 60, and 100 w.o.) quantified by qRT-PCR. 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. **P < .01. (C) Expression levels of STAT3, IKKß, and NF-?B in the stomach from stCldn18+/+ (n = 4, 4, 10, 10) and stCldn18–/– mice (n = 6, 4, 12, 16) (8, 40, 60, and 100 w.o.) quantified by qRT-PCR. 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. *P < .05; **P < .01. (D) (Left) Immunofluorescence micrographs for CCL28 (green), a lymphocyte chemoattractant and maintainer of chronic inflammation, co-stained with a-catenin (red) and DAPI (blue) in the stomach from stCldn18+/+ and stCldn18–/– mice (10, 40, 60, and 100 w.o.). CCL28 signals were clearly observed in the stomach of the stCldn18–/– mice after middle-age (40, 60, and 100 w.o.) (arrows). High-magnification images are shown in insets. Representative images from at least 3 independent experiments are shown. Scale bars = low magnification: 50 µm, high magnification: 10 µm. (Right) Expression level of CCL28 in the stomach from stCldn18+/+ (n = 8, 4, 12, 10) and stCldn18–/– (n = 10, 4, 13 16) mice (8, 40, 60, and 100 w.o.) quantified by qRT-PCR. 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. **P < .01. (E) Expression levels of CXCL5, IKKß, NF?B, and ASCL2 in the stomach from stCldn18+/+ and stCldn18–/– mice (around 50 w.o.) after POL7085 or cimetidine treatment, quantified by qRT-PCR (n: stCldn18–/– = 7, 5, 6, given saline, POL7085, and cimetidine, respectively). 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. *P < .05; **P < .01.