Fig. 1.

Caco-2 cells are resistant to cell death induced by TNF-alpha treatment. (A) Cell viability was not altered by TNF-alpha treatment. Cytotoxicity assays were conducted on TNF-alpha-treated Caco-2 cells at 10 and 100 ng/mL concentrations. Caco-2 cells were seeded at 5×10³ cells in 96-well plates and permitted to attach to plates for 2 days, after which TNF-alpha was added. After 3 days of incubation, 10 µL of color reagent was added to each well, and the cells were incubated for 1 h. The mean absorbance at 450 nm in each set of samples was measured. Shown are the mean percentages of cell survival ± S.E. of three independent experiments. (B) Downregulation of E-cadherin was noted in the TNF-alpha (10 ng/mL)-treated Caco-2 cells, but not claudin-4. E-cadherin, and claudin-4 expression levels were assessed via Western blot. Total cell lysates were prepared. (C) Downregulation of E-cadherin and altered claudin-4 translocation were noted in the TNF-alpha (10 ng/mL)-treated Caco-2 cells. Immunofluorescence staining for E-cadherin and claudin-4 was conducted on Caco-2 cells cultured on cover slips. The cells were fixed in 4% paraformaldehyde, permeabilized for 5 min with 0.1% Triton X-100, and antibodies were added at a dilution recommended by the manufacturer. Laser scanning confocal microscopy was conducted with a MRC-1024/ES confocal microscope.