Fig. 1. Progastrin dissociates AJs and TJs. (A) Treatment of the IMGE-5 cell line, which does not produce progastrin-derived peptides, for up to 4 hours with 5 nM recombinant human progastrin_6-80 did not affect the expression of ß-catenin (ß-cat), E-cadherin (E-cad), ZO-1, occludin (Occl), claudin-1 (Cld-1), p125FAK or actin, as assessed by western blotting of cell lysates. (B) Immunostaining for AJ and TJ proteins was observed using a confocal microscope as described in Materials and Methods. Series of horizontal optical sections (0.328 µm each) were collected, and images represent a merging of sections spanning the apical region of IMGE-5 cells (ten sections, 3.28 µm). Claudin-1 (Cld-1), ZO-1 and the AJ proteins E-cadherin (E-cad) and ß-catenin (ß-cat) were located in the most apical region of the lateral membrane in untreated (U) cells. Treatment with 5 nM progastrin_6-80 (PG) for 4 hours induced a partial delocalisation of all four proteins from the membrane to the cytoplasm. Bar, 7 µm. (C) Progastrin treatment also induced a time-dependent partial dissociation of the complex between occludin (white bars) and ZO-1 (black bars), as assessed by densitometric scanning of western blots of ZO-1 immunoprecipitates of cell lysates. Densitometric analysis represents the average of four experiments, and statistical significance was assessed by Student's t test. *P<0.05; **P<0.01.