Ab11197

Glutamine assay (Glutamine/Glutamate-Glo™; Cat# J8021) was purchased from Promega (Madison, WI, USA). SLC1A5 inhibitor GPNA (G6133), spermine, and spermidine were purchased from Sigma-Aldrich (St. Louis, MO, USA). Mouse monoclonal anti-MUC2 antibody (ab11197, Abcam; Cambridge, UK), Fluorescein-labeled BacTrace anti-EHEC antibody (KPL (LGC Seracare, Milford, MA, USA), secondary donkey anti-mouse AlexaFluor™ 568-labeled antibody, and 3 kDa Alexa Fluor™ 680-labeled dextran (Cat # 34681) were from Invitrogen (Waltham, MA, USA); Hoechst for DNA/nuclear staining (Thermo Fisher Scientific; Waltham, MA, USA).

Cells were seeded on Chamber slide (C6932, Sigma-Aldrich) with the seeding density of 30 × 10³ per well. The cells were allowed to attach to the wells overnight before transfecting with siRNA. After transfection with the siRNA for 72 h, slides were incubated with 4% paraformaldehyde (sc-281692, Santa Cruz, Heidelberg, Germany) 20 min at room temperature; PBS 5 min 3 times, 0.1% Triton X-100 (11332481001, Sigma-Aldrich, Saint Louis, MO, USA) 10 min at room temperature, PBS 5 min 3 times, 3% BSA block 1 h at 37 °C. After that, the slides were incubated with anti-MUC2 antibody (ab11197, Abcam, Cambridge, UK, 1:500) for 3 h at 37 °C, PBS 5 min 3 times, and with anti-mouse IgG (ab150113, Abcam, Cambridge, UK, 1:1000) for 1 h at 37 °C, PBS 5 min 3 times. The nucleus was dyed with 1 ug/mL DAPI (62248, Thermo Scientific) for 2 min at room temperature, PBS 2 min 3 times. The slides were mounted with Limonene Mounting Medium (ab104141, Abcam, Cambridge, UK,) and covered by coverslips (7695031, Th. Geyer, Renningen, Germany). Confocal microscopy was performed with Leica SP8, and images were saved as TIFF file formats. For each group in every experiment, we randomly selected three images with similar numbers of cells and uniform background.

In the present study, mouse monoclonal anti-MUC2 (ab11197, 1/500 WB and 1/100 IF, Abcam), rabbit monoclonal anti-TFF3 (ab108599, 1/1500 WB, Abcam), mouse monoclonal anti-SI ([53]; HSI-4/34 or Caco-3/73, 1/100 WB), rabbit monoclonal anti-DPPIV or CD26 [EPR20819] (ab215711, 1/2000 WB, Abcam), rabbit monoclonal anti-YAP/TAZ (D24E4, 1/1500 WB, Cell Signaling Technology, Danvers, MA, USA), rabbit monoclonal anti-YAP (D8H1X, 1/1000 WB, 1/150 IF, Cell Signaling Technology), mouse monoclonal anti-TAZ (M2-616, 1/300 IF, BD Biosciences, NJ, USA) mouse monoclonal anti-CDX2-CD88 (MU392A-UC, 1/700, BioGenex, Freemont, CA, USA), mouse monoclonal anti-HNF1α [F-7] (sc-393925, 1/300 WB, Santa Cruz Biotechnology, Dallas, TX, USA), goat polyclonal anti-HNF4α [C-19] (sc-6556, 1/600 WB, Santa Cruz Biotechnology) and anti-β-actin (MAB1501, 1/20,000, Millipore, Etobicoke, ON, Canada) were used as primary antibodies. In addition, AlexaFluor 488 or 594 goat anti-mouse (A11017, A11072, 1/400; Thermo Fisher Scientific, Ottawa, ON, Canada) and goat anti-rabbit (A11070, A11072, 1/400), ECL HRP-linked anti-mouse (NA931V, 1/4000, GE Healthcare, Mississauga, ON, Canada) and anti-rabbit (NA934V, 1/4000) and HRP-linked bovine anti-goat (sc-2350, 1/4000, Santa Cruz Biotechnology) were used as secondary antibodies.