Fig 2: Co-localization and physical interaction of CEACAM1 with ß-catenin(A and B) co-localization of E-cadherin (green, A) or ß-catenin (green, B) and CEACAM1 (red) is shown in the epithelial subpopulation of G-2 cells in immune fluorescence microscopy. (C) results from co-precipitation of CEACAM1 with ß-catenin or CDH1 from 200 µg G-2 cell lysates are shown, followed by immunoblotting with anti ß-catenin (upper panel) and anti-CEACAM1-antibodies (lower panel). Polyclonal anti-rabbit immunoglobulins or protein A/G sepharose (PAS/PGS) were used as controls. ß-catenin co-precipitates with CEACAM1 and E-cadherin, whereas precipitation of E-cadherin does not reveal binding of CEACAM1. Scale bars: 50 µm.

Fig 3: huTg rescues body weight and restores serum triglycerides in KO mice on a normal diet and high-fat diet. (A) Breeding human CEACAM1 in CEACAM1 -/- C57/BL6 mice. (B) Flow cytometric verification of human CEACAM1 expression on hepatocytes. (C) Western blot verification of human CEACAM1 expression. (D) Body weight changes (n = 6/group). (E) Serum TG levels. huTg, human CEACAM1 transgene; KO, Ceacam1 -/- ; WT, wild type [Color figure can be viewed at wileyonlinelibrary.com]

Fig 4: Analysis of body, tissue weights, survival, and serum TG of male KO vs. normal mice. (A) Weight gain (mean [SEM], n = 8) for male WT and KO mice on an ND (black symbols) and an HFD (red symbols). (B) Serum TG levels (mean [SEM]) for WT (n = 8/group) vs. KO (n = 6/group) mice on an ND and HFD. (C) Organ weights (mean [SEM], n = 3) in liver, spleen, and adipose tissues for KO vs. WT mice on an HFD. (D) Kaplan-Meier plot of survival of male WT (n = 18) vs. KO (n = 34) mice on an ND. (E) Along with mice FD, all mice at 25 months were euthanized and necropsied for presence of liver cancer, GIST, or splenomegaly. *p < 0.05, **p < 0.01, ***p < 0.001. FD, found dead; GIST, gastrointestinal stroma tumors; HFD, high-fat diet; KO, Ceacam1 -/- ; ND, normal diet; TG, triglycerides; WT, wild type [Color figure can be viewed at wileyonlinelibrary.com]

Fig 5: Loss of CEACAM1 in vivo enhances EMT and pulmonary metastasis of murine mammary adenocarcinomas(A) Quantitative evaluation of pulmonary metastasis in WAP-T and WAP-T/CEACAM1null mice presents significant differences in metastastic frequencies. Data shown in percent ± standard error (B) Histological analyses of metastasis-free lung tissue and pulmonary metastases in WAP-T and WAP-T/CEACAM1null mice reveal LT-expressing metastases in WAP-T/CEACAM1null mice (upper panel, bold arrows point at clusters of intra-vascular and inter-septal metastases). These were identified in H&E stained tissue sections of tumor-bearing WAP-T mouse (lower panels), whereas no metastases were found in the depicted tissue sections of a tumor-bearing WAP-T mouse; (C) Immune histological analyses of expression and localization of ß-catenin and ZO-1 in mammary tumors from WAP-T and WAP-T/CEACAM1null mice reveal differential localization of ß-catenin (red staining) at cell membranes (WAP-T mice, inset enlarged) and in the nuclei (WAP-T/CEACAM1null mice; insets enlarged). In immune fluorescence, ZO-1 (shown in green) is localized at cellular junctions in tumors from the WAP-T mice, but is largely absent from CEACAM1null tumors. Nuclei were stained with DAPI (blue); scale bars are indicated (D) Analyses of protein expression levels of phosphorylated ß-catenin (upper lane), and total ß-catenin (second lane), as well as total ZO-1 protein in tumor lysates from WAP-T and WAP-T/CEACAM1null mice. Protein levels were normalized to ß-Actin (lower lane). Note that phosphorylation of ß-catenin is clearly reduced in tumors from WAP-T/CEACAM1null mice. Similarly, ZO-1 protein expression is reduced in WAP-T/CEACAM1null mice. Scale bars as indicated.