Journal: Cancer Science

Article Title: Transfer RNA ‐derived fragment 5′ tRF‐Gly promotes the development of hepatocellular carcinoma by direct targeting of carcinoembryonic antigen‐related cell adhesion molecule 1

doi: 10.1111/cas.15505

Figure Lengend Snippet: Transfer RNA‐derived fragment 5′tRF‐Gly directly regulates carcinoembryonic antigen‐related cell adhesion molecule 1 (CEACAM1) expression in hepatocellular carcinoma cells. (A) Schematic illustration displaying the overlapped target genes of 5′tRF‐Gly by miRanda and RNAhybrid. Five genes were selected by Gene Ontology (GO) enrichment analysis of cell growth, cell junction, and cell–cell adhesion. (B) mRNA expression of five target genes was tested by quantitative PCR in Hep3B2.1‐7 cells after transfection with 5′tRF‐Gly mimics. (C) Protein expression of CEACAM1 was tested in Hep3B2.1‐7 cells after transfection with negative control (NC), 5′tRF‐Gly mimics, locked nucleic acid (LNA)‐NC, or LNA‐5′tRF‐Gly by western blotting. (D) Sequence of binding target of 5′tRF‐Gly in WT or mutant CEACAM1 3′‐UTR. (E) After transfection with NC or 5′tRF‐Gly mimics in 293T/17 cells, the relative luciferase activity of three kinds of mutant or WT CEACAM1 3′‐UTR was detected. (F) RIPA confirmed the binding status between 5′tRF‐Gly and CEACAM1 in untreated and treated Hep3B2.1‐7 cells. * p < 0.05; ** p < 0.01; *** p < 0.0001. n.s., not significant

Article Snippet: The Abs used in this study were rabbit anti‐CEACAM1 (ab108397; Abcam), rabbit anti‐N‐cadherin (22018‐1‐AP; Proteintech), mouse anti‐MMP2 (66366‐1‐IG; Proteintech), mouse anti‐E‐cadherin (60335‐1‐IG; Proteintech), mouse anti‐Cyclin D1 (60186‐1‐IG; Proteintech).

Techniques: Derivative Assay, Expressing, Real-time Polymerase Chain Reaction, Transfection, Negative Control, Western Blot, Sequencing, Binding Assay, Mutagenesis, Luciferase, Activity Assay

Journal: Cancer Science

Article Title: Transfer RNA ‐derived fragment 5′ tRF‐Gly promotes the development of hepatocellular carcinoma by direct targeting of carcinoembryonic antigen‐related cell adhesion molecule 1

doi: 10.1111/cas.15505

Figure Lengend Snippet: Silencing carcinoembryonic antigen‐related cell adhesion molecule 1 (CEACAM1) promotes the proliferation, migration, and invasion of hepatocellular carcinoma (HCC) cells. (A) Higher expression of CEACAM1 is related to longer progression‐free survival of HCC in the Kaplan–Meier Plotter database. (B) Expression level of CEACAM1 protein and mRNA in Hep3b2.1‐7 and Huh‐7 cells after transfection with siRNA of CEACAM1. (C) Proliferation rate of Hep3B2.1‐7 and Huh‐7 cells after CEACAM1 knockdown. (D) Representative images and quantification of Transwell migration and invasion assays after Hep3B2.1‐7 or Huh‐7 cells were transfected with negative control (siNC) or siCEACAM1. Magnification, ×100. * p < 0.05; ** p < 0.01; *** p < 0.0001

Article Snippet: The Abs used in this study were rabbit anti‐CEACAM1 (ab108397; Abcam), rabbit anti‐N‐cadherin (22018‐1‐AP; Proteintech), mouse anti‐MMP2 (66366‐1‐IG; Proteintech), mouse anti‐E‐cadherin (60335‐1‐IG; Proteintech), mouse anti‐Cyclin D1 (60186‐1‐IG; Proteintech).

Techniques: Migration, Expressing, Transfection, Negative Control

Journal: Cancer Science

Article Title: Transfer RNA ‐derived fragment 5′ tRF‐Gly promotes the development of hepatocellular carcinoma by direct targeting of carcinoembryonic antigen‐related cell adhesion molecule 1

doi: 10.1111/cas.15505

Figure Lengend Snippet: Role of transfer RNA‐derived fragment 5′tRF‐Gly on tumor progression is mediated by the downregulation of carcinoembryonic antigen‐related cell adhesion molecule 1 (CEACAM1). (A) Alteration of CEACAM1 after cotransfection with locked nucleic acid (LNA)‐5′tRF‐Gly or negative control (LNA‐NC) with either CEACAM1 or NC siRNA. (B) Proliferation of Hep3B2.1‐7 cells after cotransfection with LNA‐5′tRF‐Gly or LNA‐NC with either CEACAM1 or NC siRNA. (C, D) Representative images (C) and quantification (D) of Transwell migration and invasion assays after Hep3B2.1‐7 cells were cotransfected with LNA‐5′tRF‐Gly or LNA‐NC with either CEACAM1 or NC siRNA. (E) Protein expression of the possible downstream genes of CEACAM1 were tested in Hep3B2.1‐7 cells after cotransfection with LNA‐5′tRF‐Gly or LNA‐NC with either CEACAM1 or NC siRNA by western blot analysis. (F) Alterations of possible downstream genes of CEACAM1 in Hep3B2.1‐7 after transfection with NC, 5′tRF‐Gly mimics, LNA‐NC, or LNA‐5′tRF‐Gly. (G) Model diagram of 5′tRF‐Gly/CEACAM1 axis functioning in HCC. * p < 0.05; ** p < 0.01; *** p < 0.0001

Article Snippet: The Abs used in this study were rabbit anti‐CEACAM1 (ab108397; Abcam), rabbit anti‐N‐cadherin (22018‐1‐AP; Proteintech), mouse anti‐MMP2 (66366‐1‐IG; Proteintech), mouse anti‐E‐cadherin (60335‐1‐IG; Proteintech), mouse anti‐Cyclin D1 (60186‐1‐IG; Proteintech).

Techniques: Derivative Assay, Cotransfection, Negative Control, Migration, Expressing, Western Blot, Transfection

Journal: medRxiv

Article Title: Crypt-top and crypt-bottom colonic epithelial cell microRNA profiling reveals cell type-specific response in active and quiescent ulcerative colitis

doi: 10.1101/2022.09.25.22280336

Figure Lengend Snippet: (a) FACS of colonic epithelial cells and distribution of crypt-top CD44 - CD66 + and crypt-bottom CD44 + CD66 - epithelial cell types in inflammatory (aUC) (n=16) and non-inflammatory (qUC and HC) (n=15 and 17) colon tissues. Each dot represents a sample from the patients of the second study group. Mean ±SD of each group is represented by vertical lines. To compare the groups a nonparametric Mann–Whitney U test was performed, p* < 0.05. (b) MDS plot showing the similarity structure of the miRNA transcriptomes in crypt-top (CD66a + ) and crypt-bottom (CD44 + ) colonic epithelial cell populations in active (aUC) (n=16), quiescent UC (qUC) (n=15), and in HC (n=17) based on normalized expression values. The dots represent samples shaped by cell population. Dot colours represent condition. The centroid of ellipses corresponds to the condition group mean, the shapes are defined by covariance within the group. (c, d) Volcano plots of differentially expressed miRNAs in crypt-top (CD66a + ) and crypt-bottom (CD44 + ) colonic epithelial cell populations in active (aUC) (n=16), quiescent UC (qUC) (n=15), and HC (n=17). Colours indicate significantly (FDR < 0.05) differentially expressed miRNAs with an absolute value of log 2 FC > 1 between compared groups. (e-g) Venn diagrams representing the numbers of commonly and uniquely differentially expressed miRNAs in (e) crypt-bottom (CD44 + ) and (f) crypt-top (CD66a + ) epithelial cell populations in different UC activity, and (g) between crypt-bottom and crypt-top cells in the same condition.

Article Snippet: Antibodies used in this study were selected based on previous study and included: mouse anti-human CD326/EpCAM-FITC (clone VU-1D9, RRID: AB_2534535m Life Technologies, USA), mouse anti-human CD44-APC (clone G44-26, RRID: AB_395868, BD Biosciences, USA), mouse anti-human CD66a-PE (clone 283340, R&D Systems, USA).

Techniques: MANN-WHITNEY, Expressing, Activity Assay

Journal: medRxiv

Article Title: Crypt-top and crypt-bottom colonic epithelial cell microRNA profiling reveals cell type-specific response in active and quiescent ulcerative colitis

doi: 10.1101/2022.09.25.22280336

Figure Lengend Snippet: Top 10 overrepresented pathways within (a) and between (b) crypt-top (CD66a + ) and crypt-bottom (CD44 + ) colonic epithelial cell populations during active (aUC) (n=16), quiescent UC (qUC) (n=15) and in controls (HC) (n=17) identified by miRNA-target gene set enrichment analysis. Dot size represents the number of miRNA gene-target count in the significantly enriched (FDR < 0.05) Reactome pathways (a) and GO biological process (BP) categories (b) ; (c) a heatmap showing correlations between miRNA expression and endoscopic Mayo subscore in crypt-bottom (CD44 + ) (n=48) and crypt-top (CD66a + ) (n=48) colonic epithelial cell populations. Colour of the box represent the value of Spearman’s correlation coefficient (rho). Dots mark significant correlations (FDR < 0.05) in each cell population.

Article Snippet: Antibodies used in this study were selected based on previous study and included: mouse anti-human CD326/EpCAM-FITC (clone VU-1D9, RRID: AB_2534535m Life Technologies, USA), mouse anti-human CD44-APC (clone G44-26, RRID: AB_395868, BD Biosciences, USA), mouse anti-human CD66a-PE (clone 283340, R&D Systems, USA).

Techniques: Expressing

Journal: Hypertension

Article Title: Targeted Deletion of Murine CEACAM 1 Activates PI3K-Akt Signaling and Contributes to the Expression of (Pro)Renin Receptor via CREB Family and NF-κB Transcription Factors

doi: 10.1161/hypertensionaha.113.01324

Figure Lengend Snippet: Figure 4. (Pro)renin receptor (PRR) upregulation and activation of PI3K/Akt pathway in Cc1 small hairpin RNA (shRNA)–infected murine inner medullary collecting duct (IMCD) cells. Cells were transduced with shRNA Ceacam1 (KN) and scrambled (Sc) before analyzing PI3K p85α (A)- and Akt (B)-mediated effect on PRR expression in the presence or in the absence of LY: PI3K inhibitor LY294002 (20 µmol/L) and Akti: Akt inhibitor (2 µmol/L). PRR mRNA (A) and protein (B) levels were assessed by real-time polymerase chain reaction and Western blotting, respectively. Compartmental translocation of PI3K p85α, phospho-PI3K p85α (C) and Akt (D). Relative PI3K enzyme activity (E). Values are presented as mean±SEM relative to untreated cells; A, P<0.001; B, P<0.01; C, P<0.05. Cyto indicates cytosolic protein; LC, loading control; Mem, membranous protein; Nu, nuclear protein; and p-p85α, phosphor-PI3K p85α (Tyr 508).

Article Snippet: Mouse renal inner medullary collecting duct (IMCD) epithelial cells were subjected to small hairpin RNA (shRNA) transduction with mouse CEACAM1 and scramble lentiviral particles (Santa Cruz Biotechnology, Santa Cruz, CA) per manufacturer’s instruction.

Techniques: Activation Assay, shRNA, Infection, Transduction, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Translocation Assay, Activity Assay, Control

Journal: Hypertension

Article Title: Targeted Deletion of Murine CEACAM 1 Activates PI3K-Akt Signaling and Contributes to the Expression of (Pro)Renin Receptor via CREB Family and NF-κB Transcription Factors

doi: 10.1161/hypertensionaha.113.01324

Figure Lengend Snippet: Figure 6. Effect of carcinoembryonic antigen– related cell adhesion molecule 1 knockdown on cAMP response element (CRE)–binding protein 1 (CREB-1), activated transcription factor (ATF)-1, and ATF-2 binding to CRE and activator protein 1 (AP-1) elements in inner medullary collecting duct (IMCD) cells. A, Prediction of CRE and AP-1 sites in murine PRR promotor; (B–D) CREB-1, ATF-1, and ATF-2 bound to CRE; (E–G) CREB-1, ATF-1, and ATF-2 bound to AP-1.4; (H–J) CREB-1, ATF-1, and ATF-2 bound to AP-1.5. Values are presented as mean±SEM relative to untreated controls; A, P<0.001; B, P<0.01; C, P<0.05. KN indicates CEACAM1 knockdown cells; and Sc, scramble control.

Article Snippet: Mouse renal inner medullary collecting duct (IMCD) epithelial cells were subjected to small hairpin RNA (shRNA) transduction with mouse CEACAM1 and scramble lentiviral particles (Santa Cruz Biotechnology, Santa Cruz, CA) per manufacturer’s instruction.

Techniques: Knockdown, Binding Assay, Control

Journal: iScience

Article Title: CEACAM1 is a direct SOX10 target and inhibits melanoma immune infiltration and stemness.

doi: 10.1016/j.isci.2022.105524

Figure Lengend Snippet: Figure 3. SOX10 expression is correlated with CD8+ T-cell infiltration and CEACAM1 levels (A) In vivo growth comparison between the pBABE control (n = 5) and the pBABE-SOX10 overexpressing (n = 5) YUMM1.7 cell lines in C57 immune-competent mice. (B) Flow cytometry analysis of the CD4+ and CD8+ T-cell populations within the pBABE and pBABE-SOX10 tumors at endpoint. (C) Comparison of tumor size 24 days post injection between pBABE control (n = 4) and the pBABE-SOX10 overexpressing (n = 5) YUMM1.7 cell lines in RAG1 mice. (D) Flow analysis showing the proportion of CEACAM1 positive cells in various YUMM cell lines compared to isotype control. (E) Flow cytometric staining of SOX10 and CEACAM1 in OHRI-13 and SK-MEL28 human melanoma cell lines. Percentage of each group is indicated within their quadrant. (F) Flow cytometric staining of SOX10 and CEACAM1 in the YUMM1.1, YUMM1.7 and YUMM2.1 murine cell lines. Percentage of each group is indicated within their quadrant. Graphs show the mean G SEM. **p < 0.005 by two-tailed t test (B).

Article Snippet: Antibodies SOX10 New England BioLabs Cat# 89356; Clone D5V9L; RRID:AB_2792980 Beta-Actin Sigma-Aldrich Cat# A5316; Clone AC-74; RRID:AB_476743 Histone H3 New England BioLabs Cat# 4620; Clone D2B12; RRID:AB_1904005 Mouse: CEACAM1 BV421 BioLegend Cat# 134531; Clone Mab-CC1; RRID:AB_2687363 Human: CEACAM1 AF647 R&D Systems Cat# FAB2244R; Clone 283340 PDL-1 BV786 BD Biosciences Cat# 741014; Clone MIH5; RRID:AB_2740636

Techniques: Expressing, In Vivo, Comparison, Control, Flow Cytometry, Injection, Staining, Two Tailed Test

Journal: iScience

Article Title: CEACAM1 is a direct SOX10 target and inhibits melanoma immune infiltration and stemness.

doi: 10.1016/j.isci.2022.105524

Figure Lengend Snippet: Figure 4. SOX10 directly activates Ceacam1 on a distal enhancer region (A) YUMM1.1 cells were treated with siRNA at 200nM targeting Sox10 or a non-targeting control for 72 h and Sox10 knockdown was assessed by Immunoblot. (B) 72 h post siRNA transfection cells were isolated and stained for CEACAM1. Representative flow plots were generated with indicated percent CEACAM1+ cells. (C) qPCR was used to compare Sox10 knockdown efficiency with Ceacam1 isoform levels of the cells in (A). (D) YUMM1.7 pBABE and YUMM1.7 pBABE-SOX10 cells were collected and analyzed by qPCR for Sox10 and Ceacam1 isoforms. (E) YUMM1.7 pBABE and YUMM1.7 pBABE-SOX10 cells were isolated and stained for CEACAM1. Representative flow plots were generated with indicated percent CEACAM1+ cells. (F) Luciferase activity measurements from various Ceacam1 promoter fragments in YUMM1.1 and 2.1 cells. (G) RT-PCR of SOX10 ChIP in YUMM1.1 cells from various regions within the CEACAM1 -4135/-2785 fragment showing the fold enrichment over IgG. The red line represents the IgG control set to one. Data is representative of three independent experiments (C, D, F, and G). Graphs show the mean G SEM. *p < 0.05, **p < 0.005, ***p < 0.001, ****p < 0.0001 by one-way ANOVA (C and D) or by two-tailed t test (F).

Article Snippet: Antibodies SOX10 New England BioLabs Cat# 89356; Clone D5V9L; RRID:AB_2792980 Beta-Actin Sigma-Aldrich Cat# A5316; Clone AC-74; RRID:AB_476743 Histone H3 New England BioLabs Cat# 4620; Clone D2B12; RRID:AB_1904005 Mouse: CEACAM1 BV421 BioLegend Cat# 134531; Clone Mab-CC1; RRID:AB_2687363 Human: CEACAM1 AF647 R&D Systems Cat# FAB2244R; Clone 283340 PDL-1 BV786 BD Biosciences Cat# 741014; Clone MIH5; RRID:AB_2740636

Techniques: Control, Knockdown, Western Blot, Transfection, Isolation, Staining, Generated, Luciferase, Activity Assay, Reverse Transcription Polymerase Chain Reaction, Two Tailed Test

Journal: iScience

Article Title: CEACAM1 is a direct SOX10 target and inhibits melanoma immune infiltration and stemness.

doi: 10.1016/j.isci.2022.105524

Figure Lengend Snippet: Figure 5. CEACAM1 decreases CD8+ T-Cell infiltration and promotes tumor growth (A) Overlaid flow profiles representing the YUMM1.7 cells stably expressing CEACAM1 Isoform 1 or 4. (B) Quantitation of primary and secondary sphere formation in SOX10-deficient YUMM1.7 cells stably expressing CEACAM1 and controls. (C) Representative images of both primary and secondary spheres from the assays in (B) (Scale bar = 100mM). (D and E) In vivo growth comparison between the pBABE controls (n = 5), the CEACAM1 iso1 (n = 5) and iso4 (n = 4) expressing YUMM1.7 cell lines in NCG immune compromised mice (D) and C57 immune-competent mice (E). (F) Flow cytometry analysis of the CD4+ and CD8+ T-cell populations within the pBABE and pBABE-CEACAM1 tumors at endpoint. Data is representative of three independent experiments (B). Graphs show the mean G SEM. *p < 0.05, **p < 0.005, ***p < 0.001 by one-way ANOVA (B and F).

Article Snippet: Antibodies SOX10 New England BioLabs Cat# 89356; Clone D5V9L; RRID:AB_2792980 Beta-Actin Sigma-Aldrich Cat# A5316; Clone AC-74; RRID:AB_476743 Histone H3 New England BioLabs Cat# 4620; Clone D2B12; RRID:AB_1904005 Mouse: CEACAM1 BV421 BioLegend Cat# 134531; Clone Mab-CC1; RRID:AB_2687363 Human: CEACAM1 AF647 R&D Systems Cat# FAB2244R; Clone 283340 PDL-1 BV786 BD Biosciences Cat# 741014; Clone MIH5; RRID:AB_2740636

Techniques: Stable Transfection, Expressing, Quantitation Assay, In Vivo, Comparison, Flow Cytometry

Journal: iScience

Article Title: CEACAM1 is a direct SOX10 target and inhibits melanoma immune infiltration and stemness.

doi: 10.1016/j.isci.2022.105524

Figure Lengend Snippet: Figure 6. SOX10hi/CEACAM1+ cells show decreased CSC pool and CD8+ T-cell infiltration in tumors (A) Representative flow sorts showing the proportion of CEACAM1+ and CEACAM1-cells obtained following SOX10 expression in YUMM1.7 cells. (B) qPCR analysis for Sox10 and the Ceacam1 isoforms in sorted YUMM1.7 pBABE-SOX10 cells shown in (A). (C) In vivo growth comparison between the pBABE-SOX10 (n = 5), CEACAM1- (n = 5) and CEACAM1+ (n = 5) YUMM1.7 cell lines in C57 immune-competent mice. (D) Flow cytometry analysis of the CD4+ and CD8+ T-cell populations within the pBABE-SOX10, CEACAM1- and CEACAM1+ tumors at endpoint. (E) Quantitation of primary and secondary sphere formation in the YUMM1.7 pBABE-SOX10, CEACAM1- and CEACAM1+ cells. (F) Representative images of both primary and secondary sphere assays from (E) (scale bar = 100mM).

Article Snippet: Antibodies SOX10 New England BioLabs Cat# 89356; Clone D5V9L; RRID:AB_2792980 Beta-Actin Sigma-Aldrich Cat# A5316; Clone AC-74; RRID:AB_476743 Histone H3 New England BioLabs Cat# 4620; Clone D2B12; RRID:AB_1904005 Mouse: CEACAM1 BV421 BioLegend Cat# 134531; Clone Mab-CC1; RRID:AB_2687363 Human: CEACAM1 AF647 R&D Systems Cat# FAB2244R; Clone 283340 PDL-1 BV786 BD Biosciences Cat# 741014; Clone MIH5; RRID:AB_2740636

Techniques: Expressing, In Vivo, Comparison, Flow Cytometry, Quantitation 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: 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