Journal: Cell Death & Disease

Article Title: Autophagy-dependent secretion of ENO1 mediates chemoresistance of glioblastoma and tumor microenvironment remodeling

doi: 10.1038/s41419-025-08313-5

Figure Lengend Snippet: A Secretome profiling via 4D-FastDIA proteomics and GeneCards database screening identified secreted proteins. B Heatmap analysis of the identified secreted proteins. C Immunofluorescence (IF) staining demonstrating ENO1 colocalization with autophagy marker LC3B. Scale bars, 20 μm. D Western blot and ELISA analysis of secreted ENO1 levels in conditioned media from GBM cell lines exposed to TMZ (0–1000 μM, 48 h). E Time-course analysis (fixed 600 μM TMZ) monitoring ENO1 secretion by Western blot and ELISA. F Extracellular ENO1 quantification by ELISA and membrane integrity assessment via LDH release assay in 600 μM TMZ-treated models. G Co-immunoprecipitation (Co-IP) of GBM cell lysates using anti-ENO1 antibody, followed by immunoblotting with anti-TRIM16 and anti-SEC22B antibodies. H Western blot analysis of GAL3 and GAL8 expression in GBM cells treated with 600 μM TMZ. I IF double-labeling showing LLOMe (0.5 μM)-induced subcellular colocalization of LC3B and TRIM16. Scale bars, 20 μm. J Dose-response analysis: GBM cells treated with LLOMe (0, 0.25, 0.5, 1 μM; 24 h). Intracellular ENO1, LC3B-I-to-II conversion (Western blot), and secreted ENO1 (ELISA) were concurrently measured. K Time-course analysis (fixed 1 μM LLOMe; 0, 6, 12, 24 h): Intracellular ENO1/LC3B (Western blot) and secreted ENO1 (ELISA). L ATG5-knockdown validation: shNC/shATG5-transfected GBM cells ± TMZ (600 μM, 24 h). Immunoblotting for ATG5, secreted ENO1, and LC3B lipidation; secreted ENO1 quantified by ELISA. M Bafilomycin A1 (BafA1, 200 nM, 6 h pre-treatment) ± TMZ (600 μM, 24 h). Immunoblotting of whole-cell lysates (WCL) and conditioned media (CM) for LC3B/ENO1; secreted ENO1 by ELISA. N 3-Methyladenine (3-MA, 5 mM, 6 h pre-treatment) ± TMZ (600 μM, 24 h). Immunoblotting of WCL and CM for LC3B/ENO1; secreted ENO1 by ELISA. Data are expressed as mean ± SEM. ns not significant, *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Recombinant protein ENO1 (Cat# HY-P70260A) was purchased from MedChemExpress (MCE).

Techniques: Immunofluorescence, Staining, Marker, Western Blot, Enzyme-linked Immunosorbent Assay, Membrane, Lactate Dehydrogenase Assay, Immunoprecipitation, Co-Immunoprecipitation Assay, Expressing, Labeling, Knockdown, Biomarker Discovery, Transfection

Journal: Cell Death & Disease

Article Title: Autophagy-dependent secretion of ENO1 mediates chemoresistance of glioblastoma and tumor microenvironment remodeling

doi: 10.1038/s41419-025-08313-5

Figure Lengend Snippet: A Pan-cancer analysis of ENO1 expression. ENO1 expression in normal brain tissues and GBM tumor tissues at mRNA ( B ) and protein levels ( C ). D Heatmap analysis correlating ENO1 expression with clinicopathological features in glioma patients. Significant associations between ENO1 expression and WHO grading ( E ) or histopathological subtypes ( F ). G Kaplan-Meier overall survival (OS) analysis of TCGA-GBM patients stratified by high/low ENO1 mRNA expression. H Analysis of mRNA expression levels of ENO1 in patients with primary recurrent gliomas based on the CGGA database. I Overall survival (OS) analysis of TMZ-treated glioma patients stratified by ENO1 expression based on the CGGA-LGG + GBM database. J Representative IHC images and quantitative scores of ENO1 in paired primary/recurrent GBM specimens pre- and post-TMZ therapy. Scale bars, 50 μm. K Serum ENO1 levels quantified by ELISA across glioma WHO grades. L ELISA quantitative analysis of serum ENO1 concentrations in GBM patients pre- and post-TMZ chemotherapy. M TMZ sensitivity assays following exogenous rhENO1 (1 μg/mL) administration, measured by IC50 shifts. Data are expressed as mean ± SEM. ns not significant, *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Recombinant protein ENO1 (Cat# HY-P70260A) was purchased from MedChemExpress (MCE).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay

Journal: Cell Death & Disease

Article Title: Autophagy-dependent secretion of ENO1 mediates chemoresistance of glioblastoma and tumor microenvironment remodeling

doi: 10.1038/s41419-025-08313-5

Figure Lengend Snippet: A Western blot analysis of ENO1 knockdown efficiency in both intracellular and extracellular fractions of U87MG cells. B Western blot validation of ENO1 overexpression in LN229 cells and conditioned media (CM). Colony formation ( C ) and EdU proliferation ( D ) assays of parental LN229 cells treated with CM from ENO1-overexpressing LN229 cells. Scale bars, 100 μm. E , F Metastatic potential assessment using CM from ENO1-overexpressing cells: E Wound healing assay (scale bar = 500 μm). F Transwell migration and invasion assays (scale bar = 100 μm). G Western blot detection of intracellular ENO1 after 24 h treatment with recombinant human ENO1 (rhENO1, 1 μg/mL). H CCK-8 assay evaluating proliferation in rhENO1-treated cells (1 μg/mL). I Transwell migration/invasion of rhENO1-treated GBM cells (scale bars, 100 μm). J Scratch wound healing assay post-rhENO1 treatment (scale bars, 500 μm). Rescue experiments: Proliferation ( K , L ) and migration/invasion ( M , N ) capacities of GBM cells treated with shENO1 CM supplemented with rhENO1. Data are expressed as mean ± SEM. ns not significant, *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Recombinant protein ENO1 (Cat# HY-P70260A) was purchased from MedChemExpress (MCE).

Techniques: Western Blot, Knockdown, Biomarker Discovery, Over Expression, Wound Healing Assay, Migration, Recombinant, CCK-8 Assay

Journal: Cell Death & Disease

Article Title: Autophagy-dependent secretion of ENO1 mediates chemoresistance of glioblastoma and tumor microenvironment remodeling

doi: 10.1038/s41419-025-08313-5

Figure Lengend Snippet: A Immunoprecipitation-mass spectrometry (IP-MS) analysis of ENO1-interacting proteins in U87MG cells treated with rhENO1 (0-12 h) using ENO1-conjugated beads, with whole cell protein (WCP) and IgG as controls (silver staining shown). B Identification of ENO1-binding transmembrane proteins (TMPs) from 70-130 kDa differential bands by MS, screened against the Membranome database (containing 2364 TMPs). C Molecular docking model of ENO1-TLR4 interaction (ENO1 shown in deep blue, TLR4 in cyan; binding sites displayed as stick structures in corresponding colors). D – F Co-immunoprecipitation (Co-IP) validation of ENO1-TLR4 interaction in GBM cells: D Co-IP validated that ENO1 binds to TLR4 in shENO1 GBM cells. E Co-IP with anti-ENO1 antibody followed by immunoblotting of rhENO1-treated GBM cells with anti-ENO1 and anti-TLR4 antibodies. F Co-IP with anti-TLR4 antibody followed by immunoblotting of rhENO1-treated GBM cells with anti-ENO1 and anti-TLR4 antibodies. G Immunofluorescence analysis of ENO1 (green) and TLR4 (red) colocalization in rhENO1-stimulated GBM cells and TMZ-treated human GBM specimens. Scale bar = 10 μm. H Pearson correlation analysis of ENO1 and TLR4 mRNA expression in TCGA-GBM and CGGA-GBM datasets. Representative images and quantitative analysis of ( I ) proliferation, ( J ) colony formation, and migration/invasion ( K , L ) in GBM cells treated with or without TLR4 inhibition and cultured in rhENO1. Scale bars = 5 mm ( J ), 100 μm ( I , K ), and 500 μm ( L ). Data are expressed as mean ± SEM. ns not significant, *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Recombinant protein ENO1 (Cat# HY-P70260A) was purchased from MedChemExpress (MCE).

Techniques: Immunoprecipitation, Mass Spectrometry, Protein-Protein interactions, Silver Staining, Binding Assay, Co-Immunoprecipitation Assay, Biomarker Discovery, Western Blot, Immunofluorescence, Expressing, Migration, Inhibition, Cell Culture

Journal: Cell Death & Disease

Article Title: Autophagy-dependent secretion of ENO1 mediates chemoresistance of glioblastoma and tumor microenvironment remodeling

doi: 10.1038/s41419-025-08313-5

Figure Lengend Snippet: A Cluster analysis of differentially expressed genes (DEGs) between rhENO1-treated and control groups (n = 3 biological replicates). B Volcano plot of DEGs displaying: X-axis: log2 fold-change in gene expression; Y-axis: -log10(p-value) for statistical significance; Green dots: non-significant genes; Red/blue dots: significantly up-/down-regulated genes. C KEGG pathway analysis revealing significant association between ENO1 and PI3K- Akt signaling in GBM patient samples. D GSEA enrichment plot demonstrating PI3K- Akt pathway activation correlated with ENO1 expression from the TCGA database. E Western blot analysis of PI3K and Akt phosphorylation in rhENO1-treated GBM cells using anti-phospho-PI3K, anti-PI3K, anti-phospho- Akt, and anti- Akt antibodies. F Western blot analysis of PI3K/Akt phosphorylation in serum-starved GBM cells treated with/without rhENO1 (1 μg/mL) and PI3K inhibitor LY294002. Representative images and quantitative analysis of G colony formation, H proliferation, and migration/invasion I , J in GBM cells treated with or without PI3K inhibition LY294002 and cultured in rhENO1. Scale bars = 5 mm ( G ), 100 μm ( H, J ), and 500 μm ( I ). K Western blot analysis of PI3K/Akt phosphorylation in serum-starved GBM cells treated with/without rhENO1 (1 μg/mL) and TLR4 inhibitor TAK242 (10 μM). Data represent mean ± SEM; ns: not significant, *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Recombinant protein ENO1 (Cat# HY-P70260A) was purchased from MedChemExpress (MCE).

Techniques: Control, Gene Expression, Activation Assay, Expressing, Western Blot, Phospho-proteomics, Migration, Inhibition, Cell Culture

Journal: Cell Death & Disease

Article Title: Autophagy-dependent secretion of ENO1 mediates chemoresistance of glioblastoma and tumor microenvironment remodeling

doi: 10.1038/s41419-025-08313-5

Figure Lengend Snippet: A Principal component analysis (PCA) of metabolites and lipids detected by untargeted lipidomics in GBM cells treated with soluble recombinant ENO1 (rhENO1, 1 μg/mL) (n = 6 biological replicates). B KEGG pathway enrichment analysis of differential metabolites with VIP > 1. C Heatmap analysis of differential metabolites with VIP > 1 (n = 6 biological replicates). D Sphingomyelin (SM) was significantly upregulated in rhENO1-treated samples. E Western blot analysis of SPHK1 and ERK phosphorylation levels in serum-starved GBM cells treated with or without rhENO1 (1 μg/mL) for 6 h. F Western blot analysis using anti-phospho-SPHK1, anti-SPHK1, anti-phospho-ERK, and anti-ERK antibodies in serum-starved GBM cells treated with or without rhENO1 (1 μg/mL) and TLR4 inhibitor (TAK242, 10 μM) for 6 h. G Immunofluorescence staining of SPHK1 subcellular localization in GBM cells treated with rhENO1 (1 μg/mL) and TLR4 inhibitor (TAK242, 10 μM). Scale bar = 10 μm. H ELISA quantification of S1P levels in conditioned medium from rhENO1-treated GBM cells. I Western blot analysis of SPHK1 phosphorylation levels in GBM cells treated with increasing concentrations of SPHK1 inhibitor PF-543. J-M Representative images and quantification of J proliferation (scale bar = 100 μm), K colony formation (scale bar = 5 mm), L migration (scale bar = 100 μm), and M invasion (scale bar = 500 μm) in GBM cells treated with DMSO control, PF-543 (10 μM), or S1P (1 μM). Data are expressed as mean ± SEM. ns not significant, *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Recombinant protein ENO1 (Cat# HY-P70260A) was purchased from MedChemExpress (MCE).

Techniques: Recombinant, Western Blot, Phospho-proteomics, Immunofluorescence, Staining, Enzyme-linked Immunosorbent Assay, Migration, Control

Journal: Cell Death & Disease

Article Title: Autophagy-dependent secretion of ENO1 mediates chemoresistance of glioblastoma and tumor microenvironment remodeling

doi: 10.1038/s41419-025-08313-5

Figure Lengend Snippet: A Dimensionality reduction and clustering of single-cell RNA sequencing (scRNA-seq) and spatial transcriptomic RNA sequencing (stRNA-seq) data from GEO database. B Cluster analysis of peritumoral and tumor core regions in GBM specimens. C MIA (Microenvironment Cell Populations) analysis evaluating cell type enrichment across different clusters in tumor core versus peritumoral regions. D Spatial distribution patterns of ENO1 expression in GBM tumor core and peritumoral regions. E qRT-PCR quantification of M2-TAM markers (CD163, CD206, ARG1, IL-10) mRNA levels following rhENO1 treatment (1 μg/mL). F Immunofluorescence analysis of M2-TAM markers (CD68, CD163, CD206) subcellular localization and expression intensity after rhENO1 treatment (1 μg/mL). Scale bar = 10 μm. G Immunofluorescence staining of M2-TAM markers (CD163, CD206) in TMZ-treated co-culture system of sh-ENO1 U87MG cells with M0 macrophages. Scale bar = 10 μm. H qRT-PCR analysis of M2-TAM markers (CD163, CD206, ARG1) mRNA expression in TMZ-treated sh-ENO1 U87MG/M0 macrophage co-culture system. I Immunohistochemistry (IHC) analysis of the correlation between CD163 and ENO1 in GBM tissues. J Confocal microscopy quantification of membrane localization intensity for M2-TAM surface markers (CD163/CD206) following treatment with rhENO1 (1 μg/mL) and SPHK1 inhibitor PF-543 (10 μM). Scale bar = 10 μm. K qRT-PCR detection of M2-TAM markers (CD163, CD206, ARG1) mRNA levels under combined rhENO1 (1 μg/mL) and PF-543 (10 μM) treatment. L The levels of cytokines IL-10, CCL18, and TGF-β released in the supernatant of THP-1-derived macrophages were detected by ELISA after indicated treatment for 24 h. Data are expressed as mean ± SEM. ns not significant, *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Recombinant protein ENO1 (Cat# HY-P70260A) was purchased from MedChemExpress (MCE).

Techniques: RNA Sequencing, Expressing, Quantitative RT-PCR, Immunofluorescence, Staining, Co-Culture Assay, Immunohistochemistry, Confocal Microscopy, Membrane, Derivative Assay, Enzyme-linked Immunosorbent Assay

Journal: Cell Death & Disease

Article Title: Autophagy-dependent secretion of ENO1 mediates chemoresistance of glioblastoma and tumor microenvironment remodeling

doi: 10.1038/s41419-025-08313-5

Figure Lengend Snippet: A Experimental workflow of orthotopic glioma mouse model and treatment regimen (n = 6). B Representative in vivo bioluminescence images of Luci-GL261-bearing mice treated with:(i) saline control, (ii) TMZ monotherapy, (iii) TMZ + PF-543 (SPHK1 inhibitor), (iv) TMZ + PF-543 + TAK242 (TLR4 inhibitor) at days 7, 14, and 21 post-implantation. C , D Quantitative analysis of tumor growth using relative flux values (normalized to day 7) across treatment groups. E Kaplan-Meier survival curves showing intracranial tumor progression in the four treatment cohorts; n = 6 mice. F Representative immunohistochemical staining of ENO1, p-SPHK1, and CD163 in tumor sections from each treatment group. Scale bar, 100 μm. G Schematic diagram depicting the mechanistic basis of ENO1 mediating cellular communication between GBM cells and M2-TAMs through an autophagy-dependent secretory pathway and driving TMZ chemoresistance. Data represent mean ± SEM; ns not significant, *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Recombinant protein ENO1 (Cat# HY-P70260A) was purchased from MedChemExpress (MCE).

Techniques: In Vivo, Saline, Control, Immunohistochemical staining, Staining

Journal: The Journal of veterinary medical science

Article Title: Hexachlorophene and cuprizone induce the spongy change of the developing rat brain by different mechanisms: the role of 2', 3'-cyclic nucleotide 3'-phosphodiesterase (CNPase).

doi: 10.1292/jvms.11-0469

Figure Lengend Snippet: Fig. 1. Oligodendroglial apoptosis characterized by fragmented nuclei and condensed eosinophilic cytoplasm in the corpus callosum following CPZ dosing on days 6 (A, arrow), 8 (B, arrows), and 24 (C, arrow). Immunostaining of CNPase in the cerebral cortex in rats treated with HCP (D to F) or CPZ (G to I). Immunostaining of MBP (J to L) in the corpus striatum and caspase-3 (M to O) in the corpus callosum following CPZ treatment. CNPase reactivity is observed in the submembranous region of oligodendroglia in the controls (D), HCP-treated rats on day 5 (E), and CPZ-treated rats on day 24 (I). Strong CNPase immunostaining extends to the swollen cytoplasm of oligodendroglia in HCP-treated rats on day 12 (F) and CPZ-treated rats on days 6 (G) and 8 (H). Corpus striatum nerve fibers stain positively for MBP in the control (J) and recovered animals on day 24 (L). However, immunoreactivity in the corpus striatum decreased on day 8 (K). Caspase-3 expression by some oligodendroglia is observed on days 6 (M, arrows), 8 (N, arrows), and 24 (O, arrow). On day 8, numerous oligodendroglia are strongly positive for caspase-3 (N, arrows).

Article Snippet: Primary antibodies, their working dilutions and suppliers were as follows: mouse monoclonal antibodies against human CNPase (1:400 in dilution, Millipore, Billerica, MA, U.S.A.), human MBP (1:25 in dilution, Serotec Ltd., Oxford, England), PLP (1:200 in dilution, Millipore, Billerica, MA, U.S.A.), and a rabbit polyclonal antibody against activated caspase-3 (1:80 in dilution, Millipore, Billerica, MA, U.S.A.).

Techniques: Immunostaining, Staining, Control, Expressing