Journal: Diabetologia

Article Title: Liver-specific reconstitution of CEACAM1 reverses the metabolic abnormalities caused by its global deletion in male mice

doi: 10.1007/s00125-017-4432-y

Figure Lengend Snippet: Tissue-specific expression of the transgene. (a–b) Mouse (mCC1) and rat (rCC1) CEACAM1 protein content in intestine (Int), kidney (Kid), heart and liver were analysed by immunoblotting with polyclonal antibodies (α). Immunoblotting with α-mActin was used to normalise for loading. (c–e) Cc1+/+ (white bars), Cc1−/− (black bars) and Cc1−/−xliver+ (grey bars) mice (n = 5/genotype; 2 months old) were fasted overnight and retro-orbital blood was drawn to assess plasma insulin (c) and C-peptide (d) levels to calculate steady-state C-peptide/insulin molar ratio (e) as a measure of insulin clearance. Assays were performed in triplicate. Values are expressed as mean ± SEM; *p ≤ 0.05 vs Cc1+/+, †p ≤ 0.05 vs Cc1−/−. (f) Primary hepatocytes of Cc1+/+, Cc1−/− and Cc1−/−xliver+ mice were treated with buffer (–) or insulin (Ins) before cell-surface proteins were labelled with biotin. Proteins were immunoprecipitated with α-streptavidin beads prior to analysis by 7% SDS-PAGE and immunoblotting with antibodies against IRα and whole mouse CEACAM1 with cross-reactivity with the rat protein (CC1). Total lysates were also analysed by immunoblotting with α-mActin. For (a), (b) and (f), gels represent more than two experiments (different mice per genotype per experiment). IB, immunoblotting; IP, immunoprecipitation

Article Snippet: Western blot analysis This was achieved using 1:1000 polyclonal antibodies against phosphorylated insulin receptor beta (p-IRβ; phospho-Y1361), IR β (C18C4) (Abcam, Cambridge, MA, USA), phosphorylated Akt (p-Ser473 Akt), Akt, α-FASN (Cell Signaling, Danvers, MA, USA), custom-made rabbit polyclonal Ab2456 against the mouse CEACAM1 extracellular domain, as titrated [ 20 ], and custom-made rat CEACAM1 (αP3[Y488]) and phosphorylated CEACAM1 (α-p-CEACAM1) mouse antibodies (Bethyl Laboratories, Montgomery, TX, USA), as titrated [ 16 , 21 ]. α-Actin, α-GAPDH and α-tubulin antibodies (Santa Cruz) were used at 1:5000 dilution for normalisation.

Techniques: Expressing, Western Blot, Clinical Proteomics, Immunoprecipitation, SDS Page

Journal: Diabetologia

Article Title: Liver-specific reconstitution of CEACAM1 reverses the metabolic abnormalities caused by its global deletion in male mice

doi: 10.1007/s00125-017-4432-y

Figure Lengend Snippet: Lipid metabolism in the liver. (a–d) H&E staining in the liver of 8-month-old Cc1+/+ (a), Cc1−/− (b), L-CC1 (c) and Cc1−/−xliver+ (d) mice (n = 5/genotype). Yellow arrow points to foci of inflammatory cell infiltrates. (e) Hepatic FAO (palmitate) in fasted Cc1+/+ (white bars), Cc1−/− (black bars), L-CC1 (light grey bars) and Cc1−/−xliver+ (dark grey bars) mice (n = 5/genotype). Assays were performed in triplicate. (f) mRNA analysis of Fgf21 (performed in triplicate) in the livers of Cc1+/+ (white bars), Cc1−/− (black bars), L-CC1 (light grey bars) and Cc1−/−xliver+ (dark grey bars) mice (n = 5; 6 months of age). Values are expressed as mean ± SEM; *p ≤ 0.05 vs Cc1+/+ and †p ≤ 0.05 vs Cc1−/−; (g–j) Mice (2 months of age) were fasted overnight (white bars or ‘F’) and refed for 7 h (black bars or ‘RF’). (g, h) Analysis of plasma insulin levels (n = 6 per genotype per feeding state) (g) and Fasn mRNA expression relative to Gapdh (n = 5 per genotype per feeding state; performed in triplicate) (h). Values are expressed as mean ± SEM. (i) Western blot analysis of liver lysates was performed to assess insulin receptor protein level (α-IRβ) and phosphorylation (α-p-IRβ). Immunoblotting with α-tubulin was carried out for normalisation. Quantification of IRβ to tubulin was measured by densitometry in fasting samples. (j) Some aliquots were subjected to immunoprecipitation with α-FASN followed by immunoblotting with αp-CEACAM1 antibody (α-p-CC1). Gels represent two separate experiments performed on different mice per genotype per feeding state. (k) FASN activity was measured in triplicate by [14C]malonyl-CoA incorporation (n = 5 per genotype per feeding state). Values are expressed as mean ± SEM. For (g–i) and (k), *p < 0.05 refed vs fasted per genotype, †p ≤ 0.05 Cc1−/− vs other genotypes at fasting, ‡p ≤ 0.05 vs other genotypes at refeeding. IB, immunoblotting; IP, immunoprecipitation

Article Snippet: Western blot analysis This was achieved using 1:1000 polyclonal antibodies against phosphorylated insulin receptor beta (p-IRβ; phospho-Y1361), IR β (C18C4) (Abcam, Cambridge, MA, USA), phosphorylated Akt (p-Ser473 Akt), Akt, α-FASN (Cell Signaling, Danvers, MA, USA), custom-made rabbit polyclonal Ab2456 against the mouse CEACAM1 extracellular domain, as titrated [ 20 ], and custom-made rat CEACAM1 (αP3[Y488]) and phosphorylated CEACAM1 (α-p-CEACAM1) mouse antibodies (Bethyl Laboratories, Montgomery, TX, USA), as titrated [ 16 , 21 ]. α-Actin, α-GAPDH and α-tubulin antibodies (Santa Cruz) were used at 1:5000 dilution for normalisation.

Techniques: Staining, Clinical Proteomics, Expressing, Western Blot, Phospho-proteomics, Immunoprecipitation, Activity Assay

Journal: Diabetologia

Article Title: Liver-specific reconstitution of CEACAM1 reverses the metabolic abnormalities caused by its global deletion in male mice

doi: 10.1007/s00125-017-4432-y

Figure Lengend Snippet: Regulation of hypothalamic FASN activity. Hypothalami were extracted from the same 2-month-old mice used in Fig. 3g–k after being fasted (‘F’ or white bars) or refed for 7 h (‘RF’ or black bars). (a) Lysates were analysed by immunoblotting with α-IRβ or α-p-IRβ, using α-tubulin for normalisation. (b) mRNA analysis of Fasn relative to Gapdh (n = 5 per genotype per feeding state) was performed in triplicate. Values are expressed as mean ± SEM; †p ≤ 0.05 Cc1−/− vs other genotypes at fasting, ‡p ≤ 0.05 vs other genotypes at refeeding. (c) As in Fig. 3j, some aliquots were subjected to immunoprecipitation with α-FASN followed by immunoblotting with the α-p-CEACAM1 antibody (α-p-CC1) that also recognises CEACAM2 (a related protein with a cytoplasmic tail that shares a very high homology with that of CEACAM1). Proteins were reimmunoblotted with α-CEACAM1 antibody (α-CC1) for normalisation and with α-FASN antibody to account for the amount of immunoprecipitated FASN. Gels represent two separate experiments performed on different mice per genotype per feeding state. (d) FASN activity was measured in triplicate by [14C]malonyl-CoA incorporation (n = 5 per genotype per feeding state). Values are expressed as mean ± SEM; *p < 0.05 refed vs fasted per genotype, †p ≤ 0.05 Cc1−/− vs other genotypes at fasting, ‡p ≤ 0.05 vs other genotypes at refeeding; (e) Daily food intake (n = 6 per genotype per treatment; 6-month-old mice) and (f) body weight (BWT) (n = 6 per genotype per treatment; 6-month-old) were assessed over a period of 4 days in mice receiving an i.p. injection of vehicle (Veh, light grey bars) or C75 (dark grey bars). Data are presented as the difference between day 4 and day 0 of treatment. Values are expressed as mean ± SEM. For (e) and (f), *p < 0.05 vs vehicle-treated Cc1+/+, †p < 0.05 vs vehicle-treated within genotype. IB, immunoblotting; IP, immunoprecipitation

Article Snippet: Western blot analysis This was achieved using 1:1000 polyclonal antibodies against phosphorylated insulin receptor beta (p-IRβ; phospho-Y1361), IR β (C18C4) (Abcam, Cambridge, MA, USA), phosphorylated Akt (p-Ser473 Akt), Akt, α-FASN (Cell Signaling, Danvers, MA, USA), custom-made rabbit polyclonal Ab2456 against the mouse CEACAM1 extracellular domain, as titrated [ 20 ], and custom-made rat CEACAM1 (αP3[Y488]) and phosphorylated CEACAM1 (α-p-CEACAM1) mouse antibodies (Bethyl Laboratories, Montgomery, TX, USA), as titrated [ 16 , 21 ]. α-Actin, α-GAPDH and α-tubulin antibodies (Santa Cruz) were used at 1:5000 dilution for normalisation.

Techniques: Activity Assay, Western Blot, Immunoprecipitation, Injection

Journal: Veterinary pathology

Article Title: Morphological and immunohistochemical characterization and molecular classification of spontaneous mammary gland tumors in macropods

doi: 10.1177/03009858211069164

Figure Lengend Snippet: Primary antibodies used for IHC.

Article Snippet: EGFR , Biorbyt , 1:100 , Rabbit , CC1.

Techniques:

Journal: The Journal of Clinical Investigation

Article Title: Hepatic CEACAM1 expression indicates donor liver quality and prevents early transplantation injury

doi: 10.1172/JCI133142

Figure Lengend Snippet: Livers from groups of WT and CC1-KO C57BL/6 donor mice, stored in UW solution (4°C/18 hours), were transplanted to WT C57BL/6 recipient mice. OLT and serum samples were analyzed 6 hours after reperfusion. The sham group underwent the same procedures except for OLT. (A) Representative H&E staining (original magnification ×100). (B) Suzuki’s histological grading of liver IRI. (C) sAST and sALT levels (IU/L). (D) Representative TUNEL staining and immunohistochemical staining of OLT-infiltrating CD11b+ and Ly6G+ cells (original magnification ×200). (E) Quantification of TUNEL-positive cells/HPF. (F) Serum HMGB1 (ng/mL) and MCP1 (pg/mL) levels measured by ELISA. (G) Quantification of hepatic CD11b+ and Ly6G+ cells/HPF. (H) Real-time reverse transcription PCR–assisted (qRT-PCR–assisted) detection of mRNA coding for MCP1, CXCL1, CXCL2, and CXCL10 in OLT. Data were normalized to HPRT gene expression. Data are mean ± SD. *P < 0.05, 1-way ANOVA followed by Tukey’s HSD test (B, C, and E–G) or Student’s t test (H), n = 5–6/group.

Article Snippet: Primary Abs detecting HMGB1 (6893/D3E5), Histone H3 (4499/D1H2), cleaved caspase-3 (9664/5A1E), RIP3 (95702/D4G2A), CC1 (14771/D1P4T), ASK1 (8662/D11C9), p-p38 (4511/D3F9), Vinculin (VCL) (18799/E1E9V), and β-actin (12620/D6A8, Cell Signaling Technology) were used.

Techniques: Staining, TUNEL Assay, Immunohistochemical staining, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Expressing

Journal: The Journal of Clinical Investigation

Article Title: Hepatic CEACAM1 expression indicates donor liver quality and prevents early transplantation injury

doi: 10.1172/JCI133142

Figure Lengend Snippet: (A) Groups of WT and CC1-KO liver grafts stored in UW solution (4°C/18 hours) were perfused with physiological saline (2 mL) via a cuff placed at the portal vein to collect liver flush from inferior vena cava. (B) Liver flush samples (20 μL) from cold-stressed WT or CC1-deficient livers were screened by Western blots for HMGB1/Histone H3 levels (n = 4/group, *P < 0.05, Student’s t test). (C) WT or CC1-KO liver grafts were collected after cold storage (4°C/18 hours). Representative (n = 3/group) immunohistochemical staining of CC1/4HNE (a ROS metabolite), CC1/HMGB1, and TUNEL is shown. Arrowheads indicate extranuclear HMGB1 localization. (D) BMDM cultures (WT) were stimulated (6 hours) with liver flush obtained from WT or CC1 KO cold-stored grafts. qRT-PCR–assisted detection of mRNA coding for MCP1, CXCL2, CXCL10 with β2M normalization (n = 4–6, *P < 0.05, 1-way ANOVA followed by Tukey’s HSD test).

Article Snippet: Primary Abs detecting HMGB1 (6893/D3E5), Histone H3 (4499/D1H2), cleaved caspase-3 (9664/5A1E), RIP3 (95702/D4G2A), CC1 (14771/D1P4T), ASK1 (8662/D11C9), p-p38 (4511/D3F9), Vinculin (VCL) (18799/E1E9V), and β-actin (12620/D6A8, Cell Signaling Technology) were used.

Techniques: Western Blot, Immunohistochemical staining, Staining, TUNEL Assay, Quantitative RT-PCR

Journal: The Journal of Clinical Investigation

Article Title: Hepatic CEACAM1 expression indicates donor liver quality and prevents early transplantation injury

doi: 10.1172/JCI133142

Figure Lengend Snippet: (A) Groups of WT and CC1-KO livers were stored in UW solution (4°C/18 hours). Liver samples were collected right after cold storage (before OLT) or 3 hours after reperfusion (post-OLT). (B and C) Western blot–assisted detection and relative intensity ratio of cleaved caspase-3, RIP3, CC1, ASK1, p-p38 in naive liver, cold-stored liver or postreperfusion OLT (WT). Vinculin (VCL) expression served as an internal control and was used for normalization (n = 3/group). (D) Western blot–assisted detection and relative intensity ratio of CC1, ASK1, and p-p38 in naive WT or CC1-KO liver. VCL expression served as an internal control and was used for normalization (n = 4/group). (E) Western blot–assisted detection and relative intensity ratio of CC1, ASK1, and p-p38 in cold-stored WT or CC1-KO livers. VCL expression served as an internal control and used for normalization (n = 3/group). Data shown as mean ± SD. *P < 0.05, 1-way ANOVA followed by Tukey’s HSD test (B and C) or Student’s t test (D and E).

Article Snippet: Primary Abs detecting HMGB1 (6893/D3E5), Histone H3 (4499/D1H2), cleaved caspase-3 (9664/5A1E), RIP3 (95702/D4G2A), CC1 (14771/D1P4T), ASK1 (8662/D11C9), p-p38 (4511/D3F9), Vinculin (VCL) (18799/E1E9V), and β-actin (12620/D6A8, Cell Signaling Technology) were used.

Techniques: Western Blot, Expressing

Journal: The Journal of Clinical Investigation

Article Title: Hepatic CEACAM1 expression indicates donor liver quality and prevents early transplantation injury

doi: 10.1172/JCI133142

Figure Lengend Snippet: (A) Primary mouse hepatocytes (WT) with or without cold stimulation (4°C/4 hours) were incubated for the indicated time periods. Western blot–assisted detection and relative intensity ratio of CC1, ASK1, p-p38. VCL expression served as an internal control and used for normalization (n = 2/group). (B–D) Cold-stimulated WT or CC1-KO hepatocytes were pretreated with or without siRNA against ASK1. (B) Western blot–assisted detection and relative intensity ratio of CC1, ASK1, p-p38. VCL expression served as an internal control and used for normalization (n = 3/group). (C) Representative (n = 3/group) immunohistochemical staining of 4HNE (red, upper panels), HMGB1 (red, middle panels), and dead cell detection (red, lower panels). (D) Quantification of dead cells/HPF (n = 4–5/group). *P < 0.05, 1-way ANOVA followed by Tukey’s HSD test.

Article Snippet: Primary Abs detecting HMGB1 (6893/D3E5), Histone H3 (4499/D1H2), cleaved caspase-3 (9664/5A1E), RIP3 (95702/D4G2A), CC1 (14771/D1P4T), ASK1 (8662/D11C9), p-p38 (4511/D3F9), Vinculin (VCL) (18799/E1E9V), and β-actin (12620/D6A8, Cell Signaling Technology) were used.

Techniques: Incubation, Western Blot, Expressing, Immunohistochemical staining, Staining

Journal: The Journal of Clinical Investigation

Article Title: Hepatic CEACAM1 expression indicates donor liver quality and prevents early transplantation injury

doi: 10.1172/JCI133142

Figure Lengend Snippet: (A–C) Groups of WT and CC1-KO livers were stored in UW solution (4°C/18 hours) with or without ASK1 inhibitor (10 μg/15 mL). (A) Western blot–assisted detection and relative intensity ratio of CC1 and p-p38. VCL expression served as an internal control and used for normalization (n = 3–4/group). (B) Representative (n = 3/group) immunohistochemical staining of CC1/4HNE and CC1/HMGB1. (C) Liver flush (20 μL) from cold-stressed WT or CC1-KO livers with or without ASK1 inhibitor were analyzed by Western blots for HMGB1 levels (n = 3–4/group). (D–H) Cold-stored (4°C/18 hours) WT or CC1-KO livers were transplanted into recipient mice, and OLT and serum samples were analyzed at 6 hours after reperfusion. Some CC1-KO grafts were preincubated with ASK1 inhibitor (10 μg/15 mL) during cold storage (4°C/18 hours). Separate OLT recipient groups were monitored for 20-day survival. (D) Representative H&E (original magnification ×100) and TUNEL staining. (E) sAST and sALT levels (IU/L; n = 7–8/group). (F) Suzuki’s histological grading of liver IRI (n = 7–8/group). (G) Quantification of TUNEL-positive cells/HPF (n = 7–8/group). Data shown as mean ± SD. *P < 0.05, 1-way ANOVA followed by Tukey’s HSD test. (H) Recipient mice were monitored for 20 days and cumulative survival was analyzed (Kaplan-Meier method). Dotted line: WT → WT; solid line: CC1-KO → WT; bold line: CC1-KO+ASK1 inhibitor → WT (n = 6–9/group; *P < 0.05 vs. CC1-KO → WT, log-rank test).

Article Snippet: Primary Abs detecting HMGB1 (6893/D3E5), Histone H3 (4499/D1H2), cleaved caspase-3 (9664/5A1E), RIP3 (95702/D4G2A), CC1 (14771/D1P4T), ASK1 (8662/D11C9), p-p38 (4511/D3F9), Vinculin (VCL) (18799/E1E9V), and β-actin (12620/D6A8, Cell Signaling Technology) were used.

Techniques: Western Blot, Expressing, Immunohistochemical staining, Staining, TUNEL Assay

Journal: The Journal of Clinical Investigation

Article Title: Hepatic CEACAM1 expression indicates donor liver quality and prevents early transplantation injury

doi: 10.1172/JCI133142

Figure Lengend Snippet: Pretransplant (after cold storage) human liver Bx (n = 60) were analyzed by Western blots with β-actin normalization for CEACAM1, ASK1, and p-p38 levels (see Supplemental Figure 1A). (A) Bx samples were divided into low (n = 30) and high (n = 30) CEACAM1 expression groups based on the relative CEACAM1/β-actin levels (cutoff = 0.85, median). (B) Western blot–assisted expression of ASK1 and p-p38. Data shown in dot plots and bars indicate mean ± SEM. #P < 0.05 (Mann-Whitney U test). (C) Four representative Western blots are shown (case 1/2: low CEACAM1, case 3/4: high CEACAM1). (D) Representative (n = 3) CEACAM1/4HNE staining (original magnification ×200). (E) Representative (n = 3) CEACAM1/HMGB1 staining (original magnification ×400).

Article Snippet: Primary Abs detecting HMGB1 (6893/D3E5), Histone H3 (4499/D1H2), cleaved caspase-3 (9664/5A1E), RIP3 (95702/D4G2A), CC1 (14771/D1P4T), ASK1 (8662/D11C9), p-p38 (4511/D3F9), Vinculin (VCL) (18799/E1E9V), and β-actin (12620/D6A8, Cell Signaling Technology) were used.

Techniques: Western Blot, Expressing, MANN-WHITNEY, Staining

Journal: The Journal of Clinical Investigation

Article Title: Hepatic CEACAM1 expression indicates donor liver quality and prevents early transplantation injury

doi: 10.1172/JCI133142

Figure Lengend Snippet: Pretransplant (after cold storage) human liver Bx samples were divided into low (n = 30) and high (n = 30) CEACAM1 expression groups, based on the relative CEACAM1/β-actin levels (see Figure 6A). (A) Serum AST levels at POD1–7. (B) Serum ALT levels at POD1–7. Data are mean ± SEM. #P < 0.05 (Mann-Whitney U test). (C) Post-OLT Bx were obtained at 2 hours after reperfusion from corresponding clinical cases. Representative (n = 3) TUNEL staining (original magnification ×400). (D) Incidence of EAD (Fisher’s exact test). (E) The cumulative probability of overall graft survival. (F) The cumulative probability of rejection-free graft survival. Solid line indicates low CEACAM1; dotted line indicates high CEACAM1 human OLT patient group (Kaplan-Meier method, log-rank test).

Article Snippet: Primary Abs detecting HMGB1 (6893/D3E5), Histone H3 (4499/D1H2), cleaved caspase-3 (9664/5A1E), RIP3 (95702/D4G2A), CC1 (14771/D1P4T), ASK1 (8662/D11C9), p-p38 (4511/D3F9), Vinculin (VCL) (18799/E1E9V), and β-actin (12620/D6A8, Cell Signaling Technology) were used.

Techniques: Expressing, MANN-WHITNEY, TUNEL Assay, Staining

Journal: The Journal of Clinical Investigation

Article Title: Hepatic CEACAM1 expression indicates donor liver quality and prevents early transplantation injury

doi: 10.1172/JCI133142

Figure Lengend Snippet: Pretransplant (after cold storage) human liver Bx samples were classified into low (n = 30) and high (n = 30) CEACAM1 (CC1) expression groups (see Figure 6A for details). Post-OLT Bx were obtained at 2 hours after reperfusion from corresponding cases, followed by qRT-PCR–assisted detection of mRNA coding for TLR4, CD80, CD86, CXCL10, CD68, Cathepsin G, CD28, CD4, and IL17. Data normalized to GAPDH gene expression are shown in dot plots and bars indicative of mean ± SEM. #P < 0.05 (Mann-Whitney U test).

Article Snippet: Primary Abs detecting HMGB1 (6893/D3E5), Histone H3 (4499/D1H2), cleaved caspase-3 (9664/5A1E), RIP3 (95702/D4G2A), CC1 (14771/D1P4T), ASK1 (8662/D11C9), p-p38 (4511/D3F9), Vinculin (VCL) (18799/E1E9V), and β-actin (12620/D6A8, Cell Signaling Technology) were used.

Techniques: Expressing, Quantitative RT-PCR, MANN-WHITNEY

Journal: The Journal of Clinical Investigation

Article Title: Hepatic CEACAM1 expression indicates donor liver quality and prevents early transplantation injury

doi: 10.1172/JCI133142

Figure Lengend Snippet: Pretransplant (after cold storage) human liver Bx samples (n = 60) were analyzed by Western blots with β-actin normalization for CEACAM1 levels. (A) ROC analysis of CEACAM1/β-actin for predicting EAD. Based on a ROC curve and Youden index on the basis of best accuracy in relation to EAD incidence, the CEACAM1/β-actin cutoff value of 0.71 was determined. AUROC, area under the receiver operating characteristic curve. (B) Based on the optimal cutoff value (0.71), 60 human OLTs were classified into CEACAM1/β-actin less than 0.71 (n = 20) and CEACAM1/β-actin greater than 0.71 cases (n = 40), and the incidence of EAD was evaluated. #P < 0.05 (Fisher’s exact test). (C) Serum AST and ALT levels at POD1–7. Data are mean ± SEM. #P < 0.05 (Mann-Whitney U test). (D) Stepwise multivariate logistic regression analysis was performed to identify independent risk factors of EAD.

Article Snippet: Primary Abs detecting HMGB1 (6893/D3E5), Histone H3 (4499/D1H2), cleaved caspase-3 (9664/5A1E), RIP3 (95702/D4G2A), CC1 (14771/D1P4T), ASK1 (8662/D11C9), p-p38 (4511/D3F9), Vinculin (VCL) (18799/E1E9V), and β-actin (12620/D6A8, Cell Signaling Technology) were used.

Techniques: Western Blot, MANN-WHITNEY