agarose beads  (Vector Laboratories)

Journal: Oncogene

Article Title: POM121 O-GlcNAcylation facilitates bone metastasis in non-small cell lung cancer through enhanced c-MYC nuclear import and ECM reprogramming

doi: 10.1038/s41388-026-03687-y

Figure Lengend Snippet: A Protein levels of OGT, OGA and O-GlcNAc in bone-metastatic H460-BM subline and parental H460 cells. Blots are representative of three independent experiments. B Schematic of pulldown strategy to identify O-GlcNAc-modified proteins contributing to enhanced bone metastasis in H460-BM cells. C Left, significantly enriched ( p < 0.05) biological processes (GO analysis) for O-GlcNAc-modified proteins in H460-BM cells (top 10 pathways shown). Right, Enrichment of O-GlcNAcylation nucleoporins identified by mass spectrometry. D O-GlcNAcylation protein levels of POM121 in bone-metastatic H460-BM subline and parental H460 cells. O-GlcNAcylated proteins were pulled down using sWGA-beads. Blots are representative of three independent experiments. E H460-BM cells expressing Myc (tag)-POM121 were treated with or without OGA inhibitor Thiamet G (TMG, 10 mM, 24 h). GlcNAc (+) indicates that sWGA-beads were pre-blocked with GlcNAc (500 mM, 24 h) prior to pulldown. Immunoprecipitation (IP) and immunoblots (IB) were performed using the indicated antibodies. Data are representative of three independent experiments. F H460-BM cells expressing Myc (tag)-POM121 and mutations were subjected to sWGA-beads pulldown or anti-Myc (tag)-IP. Immunoblots identified Ser199 as an O-GlcNAc modification site. Data are representative of three independent experiments. G Mapping the O-GlcNAcylated residue Ser199 on POM121 by liquid chromatography-tandem mass spectrometry (LC-MS/MS). H , I O-GlcNAcylation of POM121 at the Ser199 site in NSCLC promotes bone metastasis in nude mouse model. H Bioluminescence imaging of nude mice four weeks after intracardiac injection of H460-BM or H460-BM POM121-S199A cells (1 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\times$$\end{document} × 10 5 cells/mouse). Data are represented as mean ± SEM; n = 6 mice/group, *** p < 0.001, unpaired t-test. I Representative H&E staining and tumor area quantifications of bone metastasis sections from mice in ( H ). Dashed lines demarcate tumor boundaries. Scale bar, 20 µm. Data are represented as mean ± SEM; **** p < 0.0001, unpaired t-test.

Article Snippet: Protein A/G magnetic beads (Thermo, cat#88802), primary antibody (TABLE S ; validated for IP), species-matched IgG antibody (TABLE S ), or Succinylated Wheat Germ Agglutinin Agarose beads (sWGA-beads, VectorLabs, cat#AL-1023S) were pre-incubated with lysis buffer at room temperature for 4 h under rotation.

Techniques: Modification, Mass Spectrometry, Expressing, Immunoprecipitation, Western Blot, Residue, Liquid Chromatography, Liquid Chromatography with Mass Spectroscopy, Imaging, Injection, Staining

Journal: Oncogene

Article Title: POM121 O-GlcNAcylation facilitates bone metastasis in non-small cell lung cancer through enhanced c-MYC nuclear import and ECM reprogramming

doi: 10.1038/s41388-026-03687-y

Figure Lengend Snippet: A Analysis of POM121 and OGT expression between parental H1299 and bone-metastatic H1299-BM sublines. Data representative of three experiments. B , C O-GlcNAcylation stabilizes POM121 in H1299-BM metastatic cells. B sWGA pulldown of O-GlcNAcylated proteins from H1299 and H1299-BM lysates, immunoblotted for POM121. C Analysis POM121 stability in H1299-BM transfected with OGT siRNAs and H1299 transfected with 3×Flag-OGT plasmid. Data representative of three experiments. D O-GlcNAcylated POM121 exhibited significantly attenuated binding affinity to TRIM21 in metastatic lineage. H1299 and H1299-BM cells treated with MG132 (25 nM, 24 h). Cell lysates IP with anti-POM121 and immunoblotted for TRIM21 and ubiquitin. Data representative of three experiments. E Enhanced nuclear c-MYC accumulation in metastatic cells. Immunoblots of c-MYC levels in the nucleoplasm and cytoplasm of H1299 cells and H1299-BM cells. HSP90 and UAP56 served as fractionation controls. Data representative of three experiments. F Left, ECM-related genes mRNA levels were analyzed by RT-qPCR between H1299 and H1299-BM cells. (mean ± SEM; n = 3; unpaired t-test). Right, Analysis of PI3K-AKT-mTOR pathway activation (p-PI3K/PI3K, p-AKT/AKT, p-mTOR/mTOR) between H1299 and H1299-BM cells. Data representative of three experiments. G Pan-cancer analysis showed that POM121 was highly expressed in NSCLC. (TCGA data; * p < 0.05, ** p < 0.01, *** p < 0.001). H High POM121 and c-MYC co-expression correlates with poor NSCLC prognosis. Kaplan-Meier overall survival analysis of 2166 NSCLC patients from TCGA, stratified by high/low mRNA expression of POM121 (log-rank test, p < 0.05). I Significantly elevated POM121 and c-MYC expression in bone metastatic lesions versus primary lung lesions of LUAD patients with bone metastasis. Data derived from the GSE225208 dataset (bulk RNA-seq; n = 20 primary lung lesions vs. n = 7 bone metastatic lesions, unpaired t -test, *** p < 0.001, **** p < 0.0001).

Article Snippet: Protein A/G magnetic beads (Thermo, cat#88802), primary antibody (TABLE S ; validated for IP), species-matched IgG antibody (TABLE S ), or Succinylated Wheat Germ Agglutinin Agarose beads (sWGA-beads, VectorLabs, cat#AL-1023S) were pre-incubated with lysis buffer at room temperature for 4 h under rotation.

Techniques: Expressing, Transfection, Plasmid Preparation, Binding Assay, Ubiquitin Proteomics, Western Blot, Fractionation, Quantitative RT-PCR, Activation Assay, Derivative Assay, RNA Sequencing