E cadherin

Caco-2 cells were cultured to confluence with mature cell-cell junctions on a collagen I coated PDMS-bottom 6-well plate (Flexcell^® vacuum-based stretching system). An equiaxial stretching of 10% constant stain was imposed for 5 min on the Caco-2 monolayer. Cells were fixed and stained for endogenous E-cadherin (mAb Rat, clone ECCD-1, Invitrogen; 191900), F-actin (Phalloidin AlexaFluor647, Invitrogen), ZO-1 (mAb mouse, Abcam; ab59720), and occludin (mAb Rabbit, Invitrogen; 6H10L9). Apical junctions of the cells were visualized and imaged by LSM 780 100× confocal microscopy. Imaging for the stretched and the control groups was conducted simultaneously with 3 independent replicative experiments. All experiment conditions were kept consistent.
E-cadherin knockdown in Caco-2 monolayer was verified by immunofluorescent staining, with E-cadherin (mAb Rabbit, Cell Signaling), α-catenin (mAb mouse, Cell Signaling), and β-catenin (mAb mouse, BD). The lysates were also immunoblotted for E-cadherin (mAb Rabbit, Cell Signaling) and GAPDH (glyceraldehyde 3-phosphate dehydrogenase) (loading control; mAb Rabbit, Sigma).

Western blotting was carried out using 40 μg of whole tissue lysate per lane with the E-cadherin antibody (1:1000, 3195, Cell signalling), β-catenin antibody (1:1000, 9582, Cell signalling), non-phospho active β-catenin antibody (1:1000, 8814S, Cell signalling), GAPDH (1:1000, ab8245, Abcam) and β-actin antibody (1:5000, A5316, Sigma). For the Atmin siRNA-mediated knockdown immunocytochemistry, E-cadherin (1:500, 3195, Cell signalling) was used.