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: Reconstitution of WT bone marrow cells in KO mice decreases Ceacam1 -/- -induced obesity. (A) Reconstitution efficiency of WT ? WT, WT ? KO, KO ? KO, and KO ? WT. (B) The body weight of chimeric mice fed a normal diet was measured at the indicated times (mean [SEM], n = 4/group). **p < 0.01, ***p < 0.001, in comparison with WT to KO. BMT, bone marrow transplantation; 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: Analyses of EMT after down-regulation of CEACAM1 expression in G-2 cells(A) CEACAM1 expression was down-regulated in stable anti-CEACAM1 shRNA –transfectants (clones G-2shCC1#2 and G-2shCC1#3); as a control, a cell clone stably expressing scrambled shRNAs was used (G-2scr). Whereas the epithelial phenotype is preserved in G-2scr cell colonies (left panel, white dotted line), the G-2shCC1#2 and G-2shCC1#3 populations exhibit a rather mesenchymal phenotype, also evident by poor intercellular adhesion and scattered cell growth (A, middle and right panel). Scale bars: 100 µm. (B) Dot plot histograms from flow cytometry analyses of the different G-2 populations are shown; they reveal maintenance of CEACAM1 expression (48.6%) in the G-2scr cells, and reduction of the fraction of CEACAM1-positive/CEACAM1high cells to 24.6% and 11.4% in the G-2shCC1#2 and G-2shCC1#3 clonal populations. (C) Comparative qRT-PCR expression analyses of key epithelial and mesenchymal marker genes (Ceacam1, Cdh1, Epcam, Cdh2, vimentin, Snai1, Twist1, Twist2, Zeb1, Tgfb1i1, Wisp1) in G-2scr, G-2shCC1#2 and G-2shCC1#3 cells. Data from G-2scr cells, G-2shCC1#2 and G-2shCC1#3 cells are shown in white, light grey and dark grey bars, respectively. Data sets are expressed as means ± SEM and were repeated three times independently in technical triplicates. Statistics: Student's t-test. (D) Comparative, representative Western blot analyses of CEACAM1, CDH1, Vimentin, Tubulin, and phosphorylated at S33/S37/T41 in ß-catenin in G-2scr, G-2shCC1#2 and G-2shCC1#3 cells. (E) Western blots of nuclear extracts showing tyrosine phosphorylation (Y86, Y654) of ß-catenin in G-2scr, G-2shCC1#2 and G-2shCC1#3 cells.