Fig. 3. Reduction of gastrin expression strengthens TJs. (A) Three independent clones of DLD-1 colorectal carcinoma cells stably transfected with an antisense gastrin construct (ASG, columns 4-6) expressed, on average, more ZO-1, occludin (Occl), E-cadherin (E-cad), claudin-1 (Cld-1), claudin-2 (Cld-2) and p125FAK (FAK) than three clones of cells transfected with vector only (VO, columns 1-3), as assessed by western blots of cell lysates. No difference was observed in the amounts of ß-catenin and actin. (B) ZO-1, occludin (Occl), claudin-1 (Cld-1) and E-cadherin (E-cad) were localised in the cytoplasm and/or nucleus in VO clones. Membrane staining for these three AJ and TJ proteins significantly increased in ASG clones, and this increase was largely reversed by a 4 hour treatment with 5 nM progastrin_6-80. Bar, 7.5 µm. (C) Treatment of ASG clones with 5 nM progastrin_6-80 for up to 240 minutes induced a partial dissociation of the complex between E-cadherin (white bars) and ß-catenin (black bars), as assessed by densitometric scanning of western blots of ß-catenin immunoprecipitates of cell lysates. Little, if any, dissociation was observed in VO clones after progastrin treatment. Densitometric analysis represents the average of at least three experiments per clone, and statistical significance was assessed by Student's t test. *P<0.05; **P<0.01.

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