Overexpression of L1 in CRC cells induces NF-κB activation and suppression of p65 expression blocks the capacity to confer liver metastasis. Overexpression of L1 in CRC cells induces NF-κB activation and suppression of p65 expression blocks the capacity to confer liver metastasis. A, the NF-κB–responsive reporter plasmid 3xκB.luc, or a control vector, was transfected together with a β-galactosidase control vector (for transfection efficiency normalization) into human HCT116 CRC cells stably overexpressing L1 (L1/Cl1 and L1/Cl2). Fold NF-κB activation was determined after dividing luciferase activity by the values obtained with the empty reporter plasmid. B, Ls174T CRC cell clones stably overexpressing L1 and shRNA to p65 (L1 + shp65/Cl1 and Cl2) or a control shRNA (L1 + control) were analyzed for L1 and p65 expression. Ponceau staining determined equal protein loading. C, the activity of NF-κB in the cell clones described in B was determined as described in A. D, the CRC cell clones (described in B) were injected into the spleen of nude mice, and tumor growth and metastasis were determined as described in Figure 1D. Nancy Gavert et al. Mol Cancer Res 2011;9:14-24 ©2011 by American Association for Cancer Research