Figure 3.

Ts-ML-ES decreases the expression of tight junction (TJ) proteins. (A) Expression of claudin-1, Occludin, and ZO-1 mRNA are altered in Caco-2 cells 48 h after treatment with Ts-ML-ES. mRNAs expression of stimulated cells (normalized with GAPDH) relative to unstimulated cells as mean ± SD from three independent experiments. ∗∗∗∗p < 0.0001 by one-way ANOVA with Tukey’s posttest. (B) The effect of Ts-ML-ES on the expression of Claudin-1, Occludin, and ZO-1 in Caco-2 cells. Fully differentiated Caco-2 cells were incubated with 25 μg/ml Ts-ML-ES (48 h). Cells without any treatments are designated as the negative control (NC) and those treated with TNF-α as the positive control (PC). β-actin was used as the internal control. Representative blots of three independent experiments are shown. (C) Effect of apical membrane exposure in control, TNF-α, or Ts-ML-ES treatment of Ts on the location of Claudin-1, Occludin, or ZO-1 in the TJs of Caco-2 cell monolayers. The merged images demonstrate the three TJ proteins in green. DNA was stained with DAPI (blue) to reveal the positions of the nuclei. (D) Transmission electron microscopy (TME) of Caco-2 cells incubated for 48 h with 20 ng/ml TNF-α, and 25 μg/ml Ts-ML-ES. The cells were then fixed with glutaraldehyde in preparation for TEM imaging. Arrowheads: Structure of TJ protein.