18Co CM increases claudin-2 expression in a KGF-dependent manner. (A) Polarized Caco-2 cells were cultured in CTL medium or 18Co CM for 24 hours, and immunofluorescence of candidate TJ proteins (claudin-1, claudin-2, occludin, and ZO-1) was examined. Fluorescent intensity of claudin-2 staining was increased by addition of 18Co CM, compared to CTL medium, without significant changes in claudin-1, occludin, or ZO-1. (B) Western blotting was performed using cell lysates from cells grown under the same conditions as in (A). 18Co CM induced an increase in claudin-2 expression without significant changes in claudin-1, occludin, and ZO-1. (C, D) Using the same culture conditions as in (A), an anti-KGF neutralizing antibody (2.5 μg/ml) was added to 18Co CM, and immunofluorescence (C) and western blot analysis (D) for claudin-2 were performed. Addition of anti-KGF neutralizing antibody to 18Co CM attenuated the 18Co CM-dependent increase in claudin-2 expression. Scale bars, 10 μm.