Fig 2: Effects of SWE on colonic ER stress markers and mucin O-glycosylation in the colon. Western blot analysis: (A) protein abundance of GRP78 and CHOP, (B) GRP78 protein expression, (C) CHOP protein expression, (D) protein abundance of C1GALT1 and Cosmc, (E) C1GALT1 protein expression. (F) Cosmc protein expression. ELISA analysis: (G) C1GALT1 level in colon tissue, (H) Cosmc level in colon tissue. Values are expressed as mean ± S.E.M. Different letters indicate significant differences (p < 0.05). n = 4–8 per group.

Fig 3: Aged distal colon shows impaired mucus barrier integrity and low level of T-synthase. (a) FISH using EUB338 probe and MUC2 antibody on distal colonic sections of mice showing bacterial penetration into the inner mucus layer. The magnified images in the 2- and 24-mo-old mouse groups showing boxed regions. The magnified image in the 16-mo-old mouse group revealing bacterial penetration of the only 16-mo-old mouse. (b) Representative fluorescent images showing UEA1 staining in the distal colons of 2-, 16-, and 24-mo-old mice. (c) Immunofluorescence staining of 2-, 16-, and 24-mo-old mouse colon sections with anti-Tn. (d) Western blot analysis showing the high expression of Tn antigen and sTn antigen in the old mouse distal mucosa compared to young. (e) T-synthase activity in the mouse distal colon and human colon. (f, g) Relative mRNA expression of C1GALT1 (f) and COSMC (g) and Western blot analysis of T-synthase (f) and Cosmc (g) protein expression in the mouse and human colon. Data are presented as mean ± SD. *** p < 0.001 compared with 2-mo-old mouse group or young people group; ## p < 0.01, ### p < 0.001 compared with 16-mo-old mouse group. Student's t test for (e-g Ms PC and Hu), and ANOVA and LSD test for (e-g Ms DC). n = 8 mice or 7 human samples per group. Abbreviations: Ms, mouse; Hu, human; PC, proximal colon; DC, distal colon.

Fig 4: Apoptosis of Tn+ cells analysis with FCM before and after treatment with WtCosmc and Apo2L/TRAIL. (A–B) Apoptosis rates in LS174T-Tn+ and LS174T-Tn- cells. (C–D) Apoptosis rates in LS174T-Tn+-Mock and LS174T-Tn+-Cosmc cells. (E–F) Apoptosis rates in Jurkat T, Jurkat T-Mock, and Jurkat T-Cosmc cells. (G) Apoptosis of LS174T-Tn- and LS174T-Tn+ cells after exposure to Apo2L/TRAIL at different concentrations (10 ng/mL, 50 ng/mL, and 100 ng/mL) before and after transfection with WtCosmc or Mock. (H) Apoptosis of Jurkat T cells after exposure to Apo2L/TRAIL at different concentrations (10 ng/mL, 20 ng/mL, and 30 ng/mL) before and after transfection with WtCosmc or Mock. (I) The apoptosis of LS174T-Tn- and LS174T-Tn+ cells were exposed to Apo2L/TRAIL at 100 ng/mL and different timepoints (6 h, 12 h, and 18 h) before and after transfection with WtCosmc or Mock. (J) Apoptosis of Jurkat T cells after exposure to Apo2L/TRAIL at 20 ng/mL and different timepoints (6 h, 12 h, and 18 h) before and after transfection with WtCosmc or Mock. Data shown are the mean {plus minus} SD of three independent experiments (***P < 0.001).

Fig 5: Transfection WtCosmc in LS174T cells and Jurkat T cells. (A) The percentage of Tn+ cells in LS174T cells and sorted LS174T-Tn+and Tn- cells by magnetic bead separation. (B) The percentage of Tn+ cells in Jurkat T cells. (C) Western blot analysis of Cosmc expression in LS174T-Tn+, LS174T-Tn+-Mock, and LS174T-Tn+-Cosmc cells. (D) Cosmc expression in Jurkat T, Jurkat T-Mock, and Jurkat T-Cosmc cells. (E) FCM analysis of the percentage of Tn+ cells in LS174T-Tn+-Mock and LS174T-Tn+-Cosmc cells. (F) The percentage of Tn+ Cells in Jurkat T-Mock and Jurkat T-Cosmc cells. (G) WtCosmc and Mock transfection cells with green fluorescence protein in LS174T-Tn+ cells as detected by immunofluorescence microscopy. (H) WtCosmc and Mock transfection cells with green fluorescence in Jurkat T cells (scale bars = 100 µm).