Figure 7

Immunofluorescence staining to localize TJ and adherens junction proteins after Caco-2 cells were incubated with IIL ESPs treated with various inhibitors in a Transwell system. Caco-2 cells were cultured on glass coverslips in a Transwell system, and 200 IIL pre-treated with various inhibitors (1.25 mM PMSF, 24 μM E-64, 2.4 mM 1,10-Phe or 10 μM pepstatin) were incubated on the insert at 37 ℃ for 2 h. Normal Caco-2 cells treated with 0.1% DMSO were used as a negative control, and normal IIL not treated with inhibitors were used to assess the ESP-mediated -hydrolysis of TJ proteins. The cells were fixed, blocked, and probed with antibodies against occludin, claudin-1, claudin-2 or E-cad followed by FITC- or Cy3-conjugated secondary antibodies. Cell nuclei were dyed blue with DAPI before examination by fluorescence microscopy (1000×). Scale bars: 50 μm.