Tff 3

Trypsin was provided by KeyGEN Biotech Co., Ltd (Nanjing, China). Dulbecco’s modified eagle medium (DMEM)-F12 (1:1) medium with high-glucose and fetal bovine serum (FBS) were purchased from Gibco/BRL (Grand Island, New York, NY, USA). 3-4,5-dimethyl-2-thiazolyl)-2,5- diphenyl-2-H-tetrazolium bromide (MTT), dimethylsulfoxide (DMSO), and 3-methyl adenine (3-MA) were provided by Sigma Chemical (St. Louis, MO, USA). Alisol A 24-acetate (24A) and Alisol B 23-acetate (23B; purity ≥95%) were purchased from Tianjin Evans Science and Technology Co., Ltd (Tianjin, China). Primary and second antibodies against LC3, Beclin-1, Bcl-2, Bcl-xl, Clusterin, Kim-1, TFF-3 and β-actin were provided by Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Hematoxylin-eosin staining solution was offered by Shanghai Yuanmu Biotechnology Co., Ltd (Shanghai, China). EliVision plus and DAB kits were offered by MXB Biological Technology Co., Ltd (Fuzhou, Fujian, China). Monopotassium phosphate, disodium hydrogen phosphate, sodium chloride, and potassium chloride were obtained from Nanjing Nanao Science and Technology Co., Ltd (Nanjing, China).

Formalin-fixed paraffin-embedded colonic tissues were sectioned (6 μm) and analyzed via immunofluorescence and FISH. For FISH analysis, tissue sections were hybridized with 10 ng/mL of a universal bacterial 16S fluorescent rRNA probe, according to previously published protocol [99 (link)]. For immunofluorescence, tissue sections were incubated with anti-CR LPS (Biotech Laboratories, Ottawa, Canada), biotinylated goat anti-GFP (GeneTex, Irvine, USA), anti-MBD-3 (Santa Cruz Biotechnology, Dallas, USA), anti-TFF3 (Santa Cruz Biotechnology, Dallas, USA) overnight at 4°C.
Confluent Caco-2 monolayers were fixed with 100% methanol and permeabilized with 0.1% Triton 1% BSA solution in PBS. Primary antibodies were used to detect β defensin-2 (Santa Cruz Biotechnology, Dallas, USA) and TFF3 (Santa Cruz Biotechnology, Dallas, USA). The selectivity of these antibodies has been previously validated [61 (link), 100 (link)–102 (link)]. Caco2 cells are known to produce low levels of TFF3, and the use of Caco2 enterocytes to detect modulation of TFF3 release has been previously validated [103 (link)–105 (link)]. Images were acquired using a Nikon epifluorescent microscope, and ImageJ was used for microscopic image analysis.