E cadherin

HTR8/SVneo, JEG3, JAR and BeWo cells were treated with trypsin and transferred into plastic tubes after cultured at a density of 2×10⁵ cells/well in 6-well microplates for 48 h. Cells were fixed in 4% paraformaldehyde for 20 min at room temperature, washed in PBS and permeabilized for 20 min in 0.1% Triton X-100-PBS. They were then washed and suspended in PBS, incubated with ST2-allophycocyanin-labeled antibodies (R&D Systems, Inc., Minneapolis, MN, USA) for 30 min at room temperature. Next, cells were washed and suspended in PBS, and immediately analyzed by four-color FCM using CellQuest Pro software, version 5.1 (FACSCalibur; BD Biosciences, Franklin Lakes, NJ, USA) with an isotypic control (BioLegend, San Diego, CA, USA).
JEG3 cells were cultured at a density of 2×10⁵ cells/well in 6-well microplates, and treated with recombinant human (rh)IL-33 (at 0 and 1 ng/ml) for 48 h, then the expression of integrin α3β1, integrin α4β1, integrin α5β1, integrin α6β1 and integrin ανβ3 (R&D Systems, Inc.), CD44, CD62L and E-cadherin (BioLegend, Inc., San Diego, CA, USA) on JEG3 cells was evaluated by FCM.

Mice were euthanized at the desired time points and the spleen, LNs, liver and lung tissues were harvested. The tissue segments were immediately washed using PBS and embedded in Tissue-Tek^® O.C.T compound (Sakura, Japan) for freezing. These samples were frozen using isopentane with dry ice and then sectioned at a thickness of 8 μm. The sections were air-dried and then fixed in acetone at –20°C for 10 min. Fixed samples were air-dried again and rehydrated using PBS containing 0.05% Tween 20 (PBST). The sections were blocked with a blocking buffer containing 10% normal donkey serum (Jackson ImmunoResearch, USA) and 1% bovine serum albumin (Sigma-Aldrich) for 1 h and then stained with the antibodies against LYVE-1, Ly5.1, B220, CD4, KLRG1, E-cadherin (Biolegend), and N-cadherin (Sigma-Aldrich). APC-conjugated streptavidin was additionally used for the detection of cells attached to a biotinylated antibody. Stained samples were washed three times using PBST and mounted with ProLong^TMAntifade Mountant (Thermo Fisher Scientific, USA). Sample images were taken using a fluorescence microscope (Axio observer.A1; Carl Zeiss, Germany). The contrast and brightness of the images were compensated and normalized using Photoshop^® CS6 (Adobe, USA) and Axio vision (Rel.4.8.2; ZEISS, USA). The number of T_M cells in the T-cell zone were counted using ImageJ software (NIH, USA).

Proteins in cells were resolved in lysis buffer which supplemented with 1 mM PMSF, and lysed for 15 min on ice. Briefly, collected the supernatant after centrifugation at 12,000×g for 10 min and diluted in 5×SDS-PAGE loading buffer (Biolegend, San Diego, CA), heated at 95 °C for 5 min and cooled on ice. Protein concentration of each sample was quantified by using of the BCA assay. Then these proteins were transferred onto the PVDF membrane (Bio-Rad, Hercules, CA) after separated. Then the membranes were blocked and reacted with first antibodies against CtBP2, E-cadherin, β-catenin, Vimentin (Biolegend, San Diego, CA) and β-actin (Abcam, Cambridge, MA) in a 4 °C refrigerator overnight. The β-actin was used for the purpose of an internal control. Following, the membranes were reacted with secondary HRP-conjugated antibody. The protein bands were visualized and imaged via chemiluminescence detection system (Tanon, Shanghai, China), and quantified by ImageQuant TL (GE healthcare life sciences, Pittsburgh, PA).