Figure 2

Claudin is exposed to the extracellular environment in non-polarized cells. (A) Model of the proposed structure of claudin proteins. The FITC-linked DFYNP peptide mimics the DFYNP sequence in the second extracellular loop of the claudin subtypes that express this conserved sequence. 21MT1 (B) and T47D (C) cells were treated with 400µM FITC-DFYNP for 1 hour at 4°C, washed 3 times with ice-cold PBS and immediately imaged live with a confocal microscope to prevent cellular uptake. Representative images show FITC-DFYNP peptide (green) bound at the surface of 21MT1 and T47D cells when treated and washed at 4°C. Cells were stained with DAPI (B&C: blue, with leakage of DAPI stain into cytosol of 21MT1 cells) and/or phalloidin (B: red). (D) FITC-peptide treated T47D cells, at 4°C, were then fixed and treated with a fluorescent antibody directed to claudin-4 (green) and stained with DAPI (blue) and phalloidin (red). Fluorescence was imaged with a confocal microscope.