Fig 2: Expression of CEACAM1-SF in CEACAM1−/−HepG2 cells and lipid droplets.A and B, flow analysis of CEACAM1 KO cells before (A) and after (B) transfection with CEACAM1-SF. C, SDS gel analysis of CEACAM1 KO cells and CEACAM1 KO cells transfected with CEACAM1-SF (4S). D, comparative lipid droplet staining for WT, CEACAM1 KO, and CEACAM1 KO cells transfected with CEACAM1-SF or CEACAM1-LF (S508A mutant). E, quantitation of lipid droplet (LD) for two sizes per cell (see Experimental procedures) is shown below each panel.

Fig 3: CEACAM1 cytoplasmic domain sequence homology and conserved functional domains. Partial sequences of rat (Rno), mouse (Mmu), and human (Has) CEACAM1 sequences taken from Kammerer and Zimmermann (28). Conserved regions for β-catenin binding (85) in blue, for ITIMs in green, and for GSK3β in red. Key residues indicated in color. Double basic residues in rat preceding Ser503 underlined.

Fig 4: siRNA-mediated silencing of CEACAM1 in ATII and A549 cells. (A) SYBR Green qRT-PCR analysis of siRNA-mediated silencing of endogenous CEACAM1-4L, -4S, -3L and -3S in ATII cells compared to cells transfected with siNeg controls. Data are expressed as fold-change (2−ΔΔCt method) normalized to β-actin and compared to mock-transfected cells that have a fold-change of 1 (dotted line). *p < 0.05, **p < 0.01. qRT-PCR was performed in duplicate and ATII cells were cultured from three donors. (B) A representative Western blot of three individual experiments and protein band density analysis of siCEACAM1-mediated knockdown of endogenous CEACAM1 in ATII and A549 cells transfected with siCEACAM1 or siNeg control or in mock-transfected cells. *p < 0.05, **p < 0.01, compared to siNeg control. Experiments were performed using ATII cells from three donors and three different passages of A549 cells. All samples on protein blots were run and cropped from the same gel for accurate standardization. Full blots are provided in the supplementary data file.

Fig 5: Effect of CEACAM1−/−and mutant cell lines on lipid droplet staining and bile canaliculi formation for HepG2 cells.A–F, lipid droplet staining with Vala (green), nuclei (blue), and F-actin (red): A, WT. B, CEACAM1−/−. C–F, CEACAM1−/− transfected with S508A mutant (C), S508D mutant (D), Y520F mutant (E), and Y493F mutant (F). G, quantitation of lipid staining of the six cell lines (see Experimental procedures). H–M, staining with anti-CEACAM1 (green), F-actin (red), and nuclei (blue). H, WT. I, KO. J–M, KO transfected with S508A mutant (J), S508D mutant (K), Y520F mutant (L), and Y493F mutant (M). Magnification 40×, arrows indicated three representative BCs located at junctions of three cells.