Journal: Cellular Oncology
Article Title: ADAMDEC1-driven CCL2-CCR2-PD-1 axis in esophageal squamous cell carcinoma: a dual threat of O-GlcNAcylation and m 6 A methylation in neoadjuvant therapy resistance and immune evasion
doi: 10.1007/s13402-026-01204-7
Figure Lengend Snippet: OGT is involved in ADAMDEC1-mediated regulation of the CCL2-CCR2-PD-1 signaling axis. ( A ) Molecular docking simulation using AlphaFold3 to predict binding modes and key amino acid interactions between ADAMDEC1 and OGT. ( B ) Immunohistochemical staining of ADAMDEC1, OGT, and RL2 (O-GlcNAcylation marker) in clinical ESCA tissues, with Pearson correlation analysis of protein expression. ( C ) Western blot detection of OGT protein and O-GlcNAc modification levels in ADAMDEC1-knockout (ADAMDEC1-KO) vs. control ESCA-PDOs. ( D ) Co-immunoprecipitation (Co-IP) assay using anti-ADAMDEC1 antibody to validate physical interaction between ADAMDEC1 and OGT in ESCA-PDOs, followed by Western blot analysis. ( E ) Cycloheximide (CHX) chase assay to measure OGT protein half-life in ADAMDEC1-KO ESCA-PDOs, with protein levels detected by Western blot at indicated time points. ( F ) Treatment of ADAMDEC1-KO ESCA-PDOs with proteasome inhibitor MG132, followed by Western blot to assess OGT protein level recovery. ( G ) Validation of ESCA-PDOs with lentiviral-mediated ADAMDEC1 knockdown and/or OGT overexpression by Western blot. ( H ) Western blot analysis of CCL2, and CCR2 protein levels in shADAMDEC1 ESCA-PDOs with OGT overexpression. ( I ) Flow cytometry detection of PD-1 expression on CD8 + T cells co-cultured with ESCA-PDOs. Un-paired Student’s t-test were used for in vivo experiments. n = 5, *** p < 0.001
Article Snippet: The protein amino acid sequences were retrieved from the UniProt database ( https://www.uniprot.org/ ) and processed through the AlphaFold3 platform ( https://deepmind.google/technologies/alphafold/alphafold-server/ ) for predicting interaction patterns.
Techniques: Binding Assay, Immunohistochemical staining, Staining, Marker, Expressing, Western Blot, Modification, Knock-Out, Control, Co-Immunoprecipitation Assay, Biomarker Discovery, Knockdown, Over Expression, Flow Cytometry, Cell Culture, In Vivo
