Claudin-1 expression increases in colon carcinoma and metastasis. (A–I) Paraffin-embedded sections of matched normal, colonic tumor, and metastatic tissues samples from the same patients were examined for claudin-1 expression and subcellular localization by immunohistochemistry using a polyclonal rabbit anti–claudin-1 antibody. (A and B) Representative normal colon tissue. Arrows indicate membranous staining. (C–E) Primary human colon adenocarcinoma tissues. Claudin-1 immunoreactivity was visible in the cytoplasm and cell nucleus (arrows). (F and G) Colon adenocarcinoma metastatic to the liver. Claudin-1 nuclear localization is shown by the arrows. (H) Tumor showing no immunoreactivity for claudin-1. (I) No staining was observed in a control experiment without primary antibody. (J) Claudin-1 expression in cell lines and human tissue samples. Equal amounts of total protein (25 μg) from various cell lines (upper panel) or tissues extracts (matched normal primary and metastasis samples; lower panel) were immunoblotted with claudin-1, claudin-7, or actin antibody. (K–P) Immunofluorescence localization of claudin-1, claudin-4, and E-cadherin in metastatic SW620 cells. (K–M) Claudin-1 (red) was predominantly localized in cell nucleus (arrows), whereas claudin-4 was largely localized on cell membrane (green; arrows). (N–P) Colocalization of claudin-1 (red) with DAPI (blue) is confirmed by overlay (purple). (Q) Cytoplasmic, nuclear, and membrane-specific fractions were prepared as described in Methods. Equal amounts of protein from all fractions were subjected to Western blot analysis using claudin-1 antibody. (R–U) Claudin-1 expression in intestinal epithelium and adenoma in ApcMin/+ mice. By immunohistochemical analysis, claudin-1 is detected in the mucosa primarily in the membrane (arrows) and in the adenoma mainly in the cytoplasm and nucleus (S–U) in ApcMin/+ mice.