Fig. 4.

Gln mitigated LPS-induced oxidative stress and S-glutathionylation of AGR2, which regulates MUC2 processing.

HT-29 CL.16E cells were cultured in the absence or presence of Gln (2 mM) and LPS (100 ng/ml) for 8 h. (A) The fluorescence intensity in HT-29 CL.16E cells was measured by flow cytometry using H2DCFDA dye (N = 6 per group). (B) The intracytoplasmic superoxide anion concentration in HT-29 CL.16E cells was assessed by flow cytometry using a dihydroethidium (DHE) probe (N = 6 per group). (C) Quantitative estimation of LPS-induced H₂O₂. Hydrogen peroxide released by cells was detected with the ROS-Glo™ Hydrogen Peroxide Kit, which was calculated by comparing the hydrogen peroxide standard curve. (D) GSH levels in cell lysates (N = 5 per group). (E) GSH/GSSG ratio in cell lysates (N = 5 per group). (F) Immunoblotting analysis of the cysteine thiol levels of AGR2 under different cellular treatments. HT-29 CL.16E cells were exposed for 30 min to H₂O₂ (200 μM), then stimulated with or without 5 mM sodium pyruvate (SP), (DTT, positive control). (G) Nonreducing electrophoresis and Western blot analysis with streptavidin-HRP to determine the S-glutathionylation level of the protein. HT-29 CL.16E cells were preloaded with BioGEE (250 μM) for 1.5 h and stimulated with H₂O₂ (200 μM) for 30 min, then cultured in the presence and absence of 5 mM sodium pyruvate (DTT, negative control). (H) Reduction electrophoresis and Western blot to determine the S-glutathionylation level of AGR2 after pulling down the biotin-GSS-protein adducts. (I) Immunoblots of AGR2 glutathionylation under different cellular treatments. HT-29 CL.16E cells were preloaded with BioGEE (250 μM) for 1.5 h, stimulated with H₂O₂ (200 μM) for 30 min, and then incubated with or without 2 mM N-acetylcysteine (DTT, negative control). (J) Western blot analysis with streptavidin-HRP to determine the S-glutathionylation level of protein. HT-29 CL.16E cells were preloaded with BioGEE (250 μM) for 1.5 h and exposed for 8 h to LPS (100 ng/ml), next cultured in the absence or presence of Gln (2 mM) and 2 mM N-acetylcysteine (DTT, negative control). (K) Immunoblotting to determine the S-glutathionylation level of AGR2 after pulling down the biotin-GSS-protein adducts. (L) Immunoblotting analysis of the cysteine thiol levels of AGR2 under different cellular treatments. HT-29 CL.16E cells were exposed for 8 h to LPS (100 ng/ml), and cultured in the absence or presence of 2 mM Gln (DTT, positive control; H₂O₂, negative control; N = 3 per group). (M) Confocal imaging of glutathionylated proteins. HT-29 CL.16E cells were cultured in the absence or presence of LPS and Gln 8 h. Green fluorescent labeling was performed using an anti-glutathionylated antibody. Blue indicates nuclear (DAPI) staining. Scale bar, 10 μm. (N) The expression of mature MUC2, immature MUC2, GRP78 and CHOP was analysed by immunoblotting. HT-29 CL.16E cells were cultured in the absence or presence of LPS and Gln (N = 3 per group). * Compared with control group, # Compared with LPS group, #P < 0.05; # #P < 0.01; # # #P < 0.001. . (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)