Journal: The Journal of Biological Chemistry

Article Title: Microbial metabolite ammonia disrupts TGF-β signaling to promote colon cancer

doi: 10.1016/j.jbc.2025.108559

Figure Lengend Snippet: High CEACAM1 levels and SMAD3 alterations are associated with significantly poorer overall survival in CRC patients. A , high CEACAM1 protein levels are observed in CRC patients. B , Kaplan–Meier survival analysis demonstrates that CRC patients with high CEACAM1 protein expression have significantly reduced overall survival compared with those with low CEACAM1 expression. C , frequent co-occurrences of increased CEACAM1 with Smad3/4 mutations are observed in CRC patients. D , Kaplan–Meier curves illustrating the overall survival in CEACAM1 high – SMAD3 MUT group (altered group) and unaltered group in TCGA CRC cohorts. Overall p value is calculated by log-rank test. Data are presented as mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001. E , structural insights reveal specific interaction sites between SPTBN1 and the CEACAM1 cytoplasmic domain. Analyses of SPTBN1 and CEACAM1 cytoplasmic domain (amino acids 453–526) interaction through AlphaFold-based molecular docking. Enlarged region shows predicted interacting residues between SPTBN1 ( orange ) and CEACAM1 ( blue ). Black arrowhead indicates the ITIM residues involved in interaction with SPTBN1. CEACAM1, carcinoembryonic antigen–related cell adhesion molecule 1; CRC, colorectal cancer; ITIM, immunoreceptor tyrosine–based inhibitory motif; SPTBN1, SMAD3 adaptor βII-spectrin; TCGA, The Cancer Genome Atlas.

Article Snippet: FL human CEACAM1 plasmid (pCMV3-SP-FLAG-CEACAM1) was purchased from Sino Biological (#HG10822-NF).

Techniques: Expressing

Journal: The Journal of Biological Chemistry

Article Title: Microbial metabolite ammonia disrupts TGF-β signaling to promote colon cancer

doi: 10.1016/j.jbc.2025.108559

Figure Lengend Snippet: CEACAM1 cytoplasmic domain contributes to ammonia-induced disruption of TGF-β signaling in human colon cancer cells. A , Western blot analyses of p-SMAD3 expression in SW480 human colon cancer cells overexpressing either full-length CEACAM1 (CEACAM1-FL) or CEACAM1 C-terminal deletion mutant (CEACAM1-ΔC). Cells were treated with NH 4 Cl (10 mM) and/or TGF-β (200 pM) as indicated. GAPDH was used as a loading control. Quantification of the p-SMAD3/SMAD3 ratio from two independent experiments. Data are presented as mean ± SD. Statistical significance was evaluated by Student’s t test. ∗ p < 0.05. B , schematic representation of CEACAM1-FL and a C-terminal deletion mutant (CEACAM1 ΔC) with individual domain information. C , schematic diagram of ( A ). CEACAM1-FL, but not CEACAM1-ΔC, interacts with SPTBN1 through its cytoplasmic domain to regulate SMAD3 phosphorylation in response to ammonia exposure, thereby disrupting TGF-β signaling. CEACAM1, carcinoembryonic antigen–related cell adhesion molecule 1; TGF-β, transforming growth factor beta.

Article Snippet: FL human CEACAM1 plasmid (pCMV3-SP-FLAG-CEACAM1) was purchased from Sino Biological (#HG10822-NF).

Techniques: Disruption, Western Blot, Expressing, Mutagenesis, Control, Phospho-proteomics

Journal: The Journal of Biological Chemistry

Article Title: Microbial metabolite ammonia disrupts TGF-β signaling to promote colon cancer

doi: 10.1016/j.jbc.2025.108559

Figure Lengend Snippet: Schematic representation of how ammonia modifies SPTBN1 cleaved products and disrupts normal SPTBN1–Smad3–TGF-β signaling to oncogenic signal. Left , normal regulation of the TGF-β pathway in intestinal epithelial cells, maintaining homeostasis. Middle , high-fat diet and other environmental toxins induces proinflammatory gut microbes, which utilize CEACAM1 for colonization, leading to an abnormal accumulation of toxic metabolites, such as ammonia. This ammonia interacts with cleaved fragments of the SMAD3/4 adaptor, SPTBN1, disrupting the normal SPTBN1–SMAD3 signaling and cause CRC. Right , our proposed therapeutic targets (SPTBN1 and CEACAM1), particularly targeting SPTBN1, can block these cleaved SPTBN1–ammonia adduct formation, thereby significantly inhibiting oncogenic signals and restoring tumor suppressor function of TGF-β signaling. CEACAM1, carcinoembryonic antigen–related cell adhesion molecule 1; CRC, colorectal cancer; SPTBN1, SMAD3 adaptor βII-spectrin; TGF-β, transforming growth factor beta.

Article Snippet: FL human CEACAM1 plasmid (pCMV3-SP-FLAG-CEACAM1) was purchased from Sino Biological (#HG10822-NF).

Techniques: Biomarker Discovery, Blocking Assay

Journal: bioRxiv

Article Title: Dynamics of the CD9 interactome during bacterial infection of epithelial cells by proximity labelling proteomics

doi: 10.1101/2024.12.13.628358

Figure Lengend Snippet: (A) CD9 knockout disrupts both meningococcal and staphylococcal adherence. WT and CD9 -/- cells were infected with either meningococci (MC58) or staphylococci (SH1000) for 60 mins at an MOI=50. Cells were disrupted after infection and adherent and internalised bacteria were enumerated by colony forming units (cfu). (B-C) CD9-derived peptide, 800C, reduces meningococcal adherence (B) and staphylcoccal adherence (C) to epithelial cells. WT or CD9 -/- cells were pre-treated with a scrambled peptide or 800C for 60 mins prior to infection. Cells were infected as above and adherence enumerated by cfu. (D) Cell surface expression of meningococcal receptors measured by flow cytometry. WT or CD9 -/- cells were treated with an anti-CD9 antibody (602.29), an anti-CD46 antibody, an anti-CD147 antibody. A mouse IgG isotype control (JC1) was included and expression was determined using a FITC-conjugated secondary antibody. % change was calculated by comparing WT to CD9 -/- cells. (E) Representative blot demonstrating expression of meningococcal and staphylococcal receptors. Whole cell lysates of WT and CD9 -/- cells were electrophoresed and blotted. Blots were probed with an anti-CD44 antibody. An anti-GAPDH antibody was used as a loading control. Densitometry was calculated through ImageJ analysis, removing background with an empty lane and normalising to the loading control. n > 3, mean + SEM.

Article Snippet: Mouse anti-human CD9 IgG1 (MM2/57; Merck, Germany), mouse anti-human CD9 IgG1 (602.29; kind gift of Lynda Partridge, University of Sheffield, Sheffield, UK), mouse anti-human CD147 IgG1 (HIM6; Biolegend, California, USA), mouse anti-human CD46 IgG1 (MEM-258; Thermo Fisher Scientific), mouse anti-human CEACAM1 IgG1 (B3-17; Merck), mouse anti-human CEACAM6 IgG1 (1H7-4B; Merck), mouse anti-human CD44 IgG2a (60224-1-Ig; Proteintech Group, Inc, Illinois, USA), mouse anti-human GAPDH (MAB374; Merck), mouse IgG1 (JC1; in house), mouse IgG2a (02-6200; Thermofisher Scientific), mouse anti-FLAG (M2; Merck), goat anti-mouse HRP (P0447; Agilent, California, USA) were used as described.

Techniques: Knock-Out, Infection, Bacteria, Derivative Assay, Expressing, Flow Cytometry, Control

Journal: Heliyon

Article Title: Establishment of novel anti-TIM-3 antibodies interfering with its binding to ligands

doi: 10.1016/j.heliyon.2024.e28126

Figure Lengend Snippet: The binding characteristics of anti-TIM-3 antibodies to ligand/TIM-3 complex. The binding of mouse anti-human TIM-3 antibodies to human Gal-9/TIM-3 (B), CEACAM-1/TIM-3 (C) or HMGB-1/TIM-3 complex (D) was detected using anti-mouse antibodies in ELISAs. Prior to being treated with mouse anti-TIM-3 mAbs, TIM-3-His were firstly attached to immobilized Gal-9, CEACAM-1, or HMGB-1 in ELISA plates. Reaction complexes were identified using anti-Ms-(H + L)-HRP. NC = negative control without TIM-3 protein; Blank = blank control without an anti-TIM-3 antibody. Horizontal lines indicate the cut-off, which were determined based on 2.1 times the mean absorbance of the negative control or blank. Statistical significance was indicated as ****p < 0.0001, ***p < 0.001, **p < 0.01, and *p < 0.05 compared with NC or Blank.

Article Snippet: Recombinant proteins, including CEACAM-1-His (Sino Biological, 10822-H08H) and HMGB-1-His (Sino Biological, 10326-H08H), were immobilized on ELISA plates at a concentration of 1 μg/mL and incubated overnight at 4 °C.

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Negative Control

Journal: Scientific reports

Article Title: Proteomic analysis of serum extracellular vesicles from biliary tract infection patients to identify novel biomarkers.

doi: 10.1038/s41598-024-56036-y

Figure Lengend Snippet: Figure 5. Validation of CEACAM1 and CRB3 expression in serum EVs. (A) CEACAM1 and CRB3 were detectable by Western blot (WB) analysis (N total = 8). (B) Densitometric quantification of CEACAM1. (C) Densitometric quantification of CRB3. (D) CRB3 was detectable by ELISA analysis. (E) CEACAM1 was detectable by ELISA analysis.* p < 0.05.

Article Snippet: Protein levels were determined using ELISA kits according to the manufacturer’s instructions: Human CEACAM1 ELISA Kit (EK1361, BOSTER, China) and Human Protein Crumbs Homolog 3 (CRB3) ELISA Kit (abx386665, abbexa, UK).

Techniques: Biomarker Discovery, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay