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: Oncology Letters

Article Title: α-enolase is highly expressed in liver cancer and promotes cancer cell invasion and metastasis

doi: 10.3892/ol.2020.12003

Figure Lengend Snippet: Comparison of the serum anti-ENO1 antibody levels among the three groups of participants [P50 (P25-P75)].

Article Snippet: The anti-ENO1 antibody ELISA detection reagent kit (cat. no. CSB-EQ027775HU) was purchased from CUSABIO Technology LLC.

Techniques: Comparison, Control

Journal: Diagnostics

Article Title: Overexpressed Proteins in HCC Cell-Derived Exosomes, CCT8, and Cofilin-1 Are Potential Biomarkers for Patients with HCC

doi: 10.3390/diagnostics11071221

Figure Lengend Snippet: Diagnostic efficiency of annexin V, CCT8, CFL1, ENO1, HSPB1, and TPM4 in diagnosing HCC in the test cohort comprised of normal healthy individuals (normal) and patients with HCC. ( a ) Comparison of the six protein expressions between 14 healthy individuals (normal) and 15 patients with HCC. ( b ) Area under the curve (AUC) and receiver operating characteristics (ROC) of six protein markers in diagnosing HCC. ( c ) Left panel: relative expression of CCT8 and CFL1 in serum exosomal mRNA of 29 healthy individuals (normal) and 20 patients with HCC. Right panel: area under the curve (AUC) and receiver operating characteristics (ROC) of serum exosomal CCT8 and CFL1 expression.

Article Snippet: The following commercially available ELISA kits were used: enolase 1 (ENO1) (CSB-E17177h; Cusabio, Houston, TX, USA), CCT8 (MBS2516261), CFL1 (MBS2505977), ANXA5 (MBS704883), HSPB1 (MBS727021), and TPM4 (MBS944560; all previously named kits from Mybiosource Inc., San Diego, CA, USA).

Techniques: Diagnostic Assay, Comparison, Expressing

Journal: Frontiers in oncology

Article Title: A Novel Invadopodia-Specific Marker for Invasive and Pro-Metastatic Cancer Stem Cells.

doi: 10.3389/fonc.2021.638311

Figure Lengend Snippet: FIGURE 1 | The expression of ENO1 on the cell-surface of a subpopulation of CSCs. (A) Representative FACS plots showing patterns of CD44, CD133, and surface ENO1 (sENO1) staining of primary prostate adenocarcinoma (PAC)-derived 22Rv-1 cells with the frequency of the boxed CD44+CD133+ cell population (representing CSCs in PAC; left) or sENO1+ cells in CD44+CD133+ CSCs (middle) or cells in the other subpopulations (representing non-CSCs; right) shown. (B) The percentages of sENO1+ cell subpopulation in CD44+CD133+ 22Rv-1 cells or cells in the other subpopulations (others). (C) The percentages of sENO1+ cell subpopulation in CD44+CD133+ PC-3 cells or cells in the other subpopulations. (D) The percentages of sENO1+ cell subpopulation in CD90+ gastric adenocarcinoma (GAC) AGS or NCI-N87 cells (representing CSCs in GAC) or CD90- cells (representing non-CSCs). Error bars represent mean ± SEM from three independent experiment (n = 3). Unpaired t-test was performed throughout where **p < 0.01; ***p < 0.001 in (B–D).

Article Snippet: In other experiments, to profile the expression pattern of sENO1 on invadopodia, the cells seeded on gelatin were immunostained for sENO1 with rabbit polyclonal anti-ENO1 (OriGene, Rockville, MD), after which the cells were fixed with 4% formaldehyde and then immunostained for cortactin and F-actin as described above.

Techniques: Expressing, Staining, Derivative Assay

Journal: Frontiers in oncology

Article Title: A Novel Invadopodia-Specific Marker for Invasive and Pro-Metastatic Cancer Stem Cells.

doi: 10.3389/fonc.2021.638311

Figure Lengend Snippet: FIGURE 2 | sENO1 marks a subpopulation of mesenchymal-like and highly invasive CSCs. (A) The relative transcript levels of the mesenchymal- (left) or pluripotency- (right) associated genes in sENO1+ CSCs (represented by CD44+CD133+ 22Rv-1 cells), sENO1- CSCs, and non-CSCs (represented by cells in the other subpopulations) using qRT-PCR analysis. Error bars represent mean ± SEM from three independent experiments (n = 3). Unpaired t-test was performed throughout where *p < 0.05 versus non-CSCs; †p < 0.05 versus sENO1- CSCs. (B) Immunoblotting analysis of the indicated markers selected from (A) in non- CSCs, sENO+, and sENO- CSCs. Protein levels were quantified by densitometric analysis of the bands, normalized to b-tubulin (loading control). (C) Limiting dilution assay (LDA) demonstrating the tumorsphere-forming efficacy of each subset of tumor cells. Three independent experiments were performed (n = 6). Shown are maximum likelihood estimates with a 95% confidence interval, where **p < 0.01. (D) The invasive capacities of freshly sorted sENO1+ CSCs (represented by CD44+CD133+ 22Rv-1 cells), sENO1- CSCs, and non-CSCs in 22Rv-1 cells in a dual-chamber invasion assay. Shown are representative immunofluorescence images of the invaded cells, with cell nuclei stained with SYTOX-green (green). Scale bars = 500 µm. Right, the number of invaded cells. Error bars represent mean ± SEM from three independent experiments (n = 3). Unpaired t-test was performed throughout where **p < 0.01.

Article Snippet: In other experiments, to profile the expression pattern of sENO1 on invadopodia, the cells seeded on gelatin were immunostained for sENO1 with rabbit polyclonal anti-ENO1 (OriGene, Rockville, MD), after which the cells were fixed with 4% formaldehyde and then immunostained for cortactin and F-actin as described above.

Techniques: Quantitative RT-PCR, Western Blot, Control, Limiting Dilution Assay, Invasion Assay, Staining

Journal: Frontiers in oncology

Article Title: A Novel Invadopodia-Specific Marker for Invasive and Pro-Metastatic Cancer Stem Cells.

doi: 10.3389/fonc.2021.638311

Figure Lengend Snippet: FIGURE 3 | sENO1+ CSCs are highly pro-metastatic. (A) Representative BLI of NOD/SCID mice receiving an intra-splenic injection of sENO1+ CSCs (represented by CD90+ NCI-N87 cells), sENO1- CSCs (CD90- NCI-N87 cells), and non-CSCs (represented by CD90- cells). at the indicated time following cell inoculation. (B) Tumor bulk quantified as BLI normalized photon counts as a function of time. Error bars represent mean ± SEM from one experiment (n = 8 mice per group). Unpaired t-test was performed throughout where **p < 0.01 versus non-CSCs. (C) Representative BLI of NOD/SCID mice receiving intra-splenic injection of sENO1+

Article Snippet: In other experiments, to profile the expression pattern of sENO1 on invadopodia, the cells seeded on gelatin were immunostained for sENO1 with rabbit polyclonal anti-ENO1 (OriGene, Rockville, MD), after which the cells were fixed with 4% formaldehyde and then immunostained for cortactin and F-actin as described above.

Techniques: Injection

Journal: Frontiers in oncology

Article Title: A Novel Invadopodia-Specific Marker for Invasive and Pro-Metastatic Cancer Stem Cells.

doi: 10.3389/fonc.2021.638311

Figure Lengend Snippet: FIGURE 4 | ENO1 is expressed on the invadopodial surface of CSCs. (A) Confocal views of PAC CSCs (represented by CD44+CD133+ PC-3 cells) showing the cross-section of invadopodia structures (represented by cortactin+F-acin+ puncta) with the colocalized surface ENO1 (sENO1; green), cortactin (red), and F-actin (magenta) that penetrate into the underlying gelatin matrix. Nuclei were counterstained with 4’,6-diamidino-2-phenylindole (DAPI; blue). Scale, 10 µm. (B) Top, a pie chart showing the percentage of sENO1+ invadopodia per PC-3 CSC. Bottom, a pie chart showing the percentage of sENO1+ invadopodia per GAC AGS CSC (represented by CD90+ AGS cells). (C) Left, representative three-dimensional (3D) reconstructed confocal image of CD44+CD133+ PC-3 CSCs showing the co- localization of sENO1 (green) and cortactin (red) at the ventral side of cell. Scale, 8 µm. Right upper, digital zoom-in image from serial Z sections (yellow rectangle) showing the spatial colocalization of sENO1 (green) and cortactin (red) at invadopodia. Scale, 5 µm. Right lower, the orthogonal view of the magnified areas (yellow squares at top) shown the distribution and localization of sENO1 and cortactin at the base of invadopodia. 3D rendered images of the invadopodia (arrows) were processed by using Imaris software. Scale, 1 µm.

Article Snippet: In other experiments, to profile the expression pattern of sENO1 on invadopodia, the cells seeded on gelatin were immunostained for sENO1 with rabbit polyclonal anti-ENO1 (OriGene, Rockville, MD), after which the cells were fixed with 4% formaldehyde and then immunostained for cortactin and F-actin as described above.

Techniques: Software

Journal: Frontiers in oncology

Article Title: A Novel Invadopodia-Specific Marker for Invasive and Pro-Metastatic Cancer Stem Cells.

doi: 10.3389/fonc.2021.638311

Figure Lengend Snippet: FIGURE 5 | sENO1+ CSCs generate more invadopodia than their sENO1- counterparts. (A) Confocal views of sENO1+ PC-3 CSCs (represented by CD44+CD133+

Article Snippet: In other experiments, to profile the expression pattern of sENO1 on invadopodia, the cells seeded on gelatin were immunostained for sENO1 with rabbit polyclonal anti-ENO1 (OriGene, Rockville, MD), after which the cells were fixed with 4% formaldehyde and then immunostained for cortactin and F-actin as described above.

Techniques:

Journal: Frontiers in oncology

Article Title: A Novel Invadopodia-Specific Marker for Invasive and Pro-Metastatic Cancer Stem Cells.

doi: 10.3389/fonc.2021.638311

Figure Lengend Snippet: FIGURE 6 | sENO1 contributes to the invadopodial formation and the matrix-degradative function of CSCs. (A) Immunoblotting analysis showing the effect of lentivirus shRNA-mediated knockdown (KD) of ENO1 expression in PC-3 cells. Protein levels were quantified by densitometric analysis of the bands, normalized to b- tubulin (loading control). (B) Bar graph showing the percentage of sENO1+ PC-3 cells with KD of ENO1 expression or control KD. (C) Bar graph showing the density of invadopodia (represented by cortactin+F-actin+ puncta) per cells in PC-3 CSCs (represented by CD44+CD133+ cells) or non-CSCs (represented by cells in other subpopulations) with ENO1 KD or control KD. Error bars represent mean ± SEM from three independent experiments (n = 3). Unpaired t-test was performed where **p < 0.01, ***p < 0.001 in (B, C). (D) PC-3 cells with KD of ENO1 expression or the control KD cells were seeded on top of a fluorescein-conjugated gelatin matrix and immunostained with cortactin (green) or phalloidin (F-actin; red). Nuclei were counterstained with DAPI (blue). Right, the fluorescence intensity of fluorescein- conjugated gelatin within the boundary (determined by F-actin staining) of PC-3 cells with ENO1 KD or control KD (n = 50 cells counted per sample). Unpaired t-test was performed where ***p < 0.001. (E) Bar graph showing the invasive capacity of PC-3 CSCs with ENO1 KD or control KD in a dual-chamber invasion assay. Error bars represent mean ± SEM from three independent experiments (n = 3). Unpaired t-test was performed where **p < 0.01 versus non-CSCs. (F) Representative immunofluorescence images of CD44+CD133+ PC-3 cells (representing CSCs) that had invaded the type I collagen matrix in the presence of an increasing concentration (0.1-1.0 µg/ml) of the anti-ENO1 polyclonal antibody (pAb; a-ENO1) in a dual-chamber invasion assay. The nuclei of the invaded cells were stained with SYTOX-green. Scale bars, 500 µm. Right, the number of invaded cells. Cells in other subpopulations (representing non-CSCs) were included as a control. Error bars represent mean ± SEM from three independent experiments (n = 3). Unpaired t-test was performed throughout where *p < 0.05, **p < 0.01 versus non-CSCs. (G) The invadopodia density per cell in PC-3 CSCs or non-CSCs exposed to an increasing concentration of a-ENO1. Error bars represent mean ± SEM from three independent experiments (n = 50 cells counted per sample). Unpaired t-test was performed where *p < 0.05, **p < 0.01, ***p < 0.001 versus non-CSCs. (H) PC-3 CSCs were seeded on top of a gelatin matrix in the presence or absence of a-ENO1 (20 µg/ml). Shown are the extent of matrix degradation as reflected by immunostaining with anti-Col1-3/4C (red). Right, the total cell fluorescence intensity of Col1-3/4C in PC-3 CSCs treated with a-ENO1 or a control IgG (n = 50 cells counted per sample). Unpaired t-test was performed where ***p < 0.001.

Article Snippet: In other experiments, to profile the expression pattern of sENO1 on invadopodia, the cells seeded on gelatin were immunostained for sENO1 with rabbit polyclonal anti-ENO1 (OriGene, Rockville, MD), after which the cells were fixed with 4% formaldehyde and then immunostained for cortactin and F-actin as described above.

Techniques: Western Blot, shRNA, Knockdown, Expressing, Control, Staining, Invasion Assay, Concentration Assay, Immunostaining

Journal: Frontiers in oncology

Article Title: A Novel Invadopodia-Specific Marker for Invasive and Pro-Metastatic Cancer Stem Cells.

doi: 10.3389/fonc.2021.638311

Figure Lengend Snippet: FIGURE 7 | CAV1 is indispensable for the surface localization of sENO1 on CSCs and its pro-invadopodia and pro-invasive functions. (A) Immunoblotting analysis showing the effect of lentivirus shRNA-mediated knockdown (KD) of CAV1 (top) or HSP70 (bottom) expression in PC-3 cells. Protein levels were quantified by densitometric analysis of the bands, normalized to b-tubulin (loading control). (B) Bar graph showing the percentage of sENO1+ cells in PC-3 CSCs (represented by CD44+CD33+ cells) with KD of CAV1 or HSP70 expression or control-KD. Unpaired t-test was performed throughout where ***p < 0.001 versus control KD. (C) Bar graph showing the density of invadopodia (represented by coractin+F-actin+ puncta) per cell in PC-3 CSCs or non-CSCs (represented by cells in other subpopulations) with CAV1 KD or control KD. Unpaired t-test was performed throughout where ***p < 0.001. (D) Bar graph showing the invasive capacity of PC-3 CSCs or non-CSCs with CAV1 KD or control KD in a dual-chamber invasion assay. Error bars represent mean ± SEM from three independent experiments (n = 3). Unpaired t-test was performed throughout where *p < 0.05 versus non-CSCs; †p < 0.05 versus control KD.

Article Snippet: In other experiments, to profile the expression pattern of sENO1 on invadopodia, the cells seeded on gelatin were immunostained for sENO1 with rabbit polyclonal anti-ENO1 (OriGene, Rockville, MD), after which the cells were fixed with 4% formaldehyde and then immunostained for cortactin and F-actin as described above.

Techniques: Western Blot, shRNA, Knockdown, Expressing, Control, Invasion Assay

Journal: International Journal of Molecular Sciences

Article Title: ENO1 Promotes OSCC Migration and Invasion by Orchestrating IL-6 Secretion from Macrophages via a Positive Feedback Loop

doi: 10.3390/ijms24010737

Figure Lengend Snippet: Expression and secretion of ENO1 in tumor cells and its regulation of lactic acid release. ( A ) Relative mRNA expression levels of ENO1 were detected by RT-qPCR in different cell lines. β-actin was used as a reference to normalize the data. ( B , C ) Relative protein expression levels of ENO1 were detected by Western blot. β-actin was used as a reference to normalize the data. ( D ) Protein secretion levels of ENO1 in cell culture supernatant were detected by ELISA. ( E ) The transfection efficiency of ENO1 mRNA expression levels were detected by RT-qPCR (48 h). ( F , G ) The transfection efficiency of ENO1 protein expression levels were detected by Western blot (72 h). ( H ) The lactic acid concentration was determined in the medium of untreated (control), scrambled siRNA and ENO1-siRNA-transfected CAL27 cells. β-actin was used as a reference to normalize the data. Different symbols (circle/square/triangle) were used to represent the data points of independent biological repeated experiments. All data are displayed as mean ± SEM; n = 3; ns, no significance, * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001. Different symbols (circle/square/triangle) were used to represent the data points of independent biological repeated experiments.

Article Snippet: The levels of human ENO1 and murine IL-6 in the cell culture supernatants were measured by ELISA kits (Cusabio, Wuhan, China), following the manufacturer’s instructions.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Cell Culture, Enzyme-linked Immunosorbent Assay, Transfection, Concentration Assay, Control

Journal: International Journal of Molecular Sciences

Article Title: ENO1 Promotes OSCC Migration and Invasion by Orchestrating IL-6 Secretion from Macrophages via a Positive Feedback Loop

doi: 10.3390/ijms24010737

Figure Lengend Snippet: ENO1 promotes tumor cell migration and invasion through macrophages. ( A , C ) Wound-healing assay of CAL27 cells incubated with Macro-CM from macrophages induced by TCM of transfected CAL27 cells. ( A ) representative pictures; ( C ) bar charts indicating the wound healing rate. Magnification 40×. Scale bar: 500 μm. ( B , D , E ) Transwell assay for migration (upper panel) and invasion (lower panel) of CAL27 cells cocultured with macrophages induced by TCM of transfected CAL27 cells. ( B ) representative pictures; ( D , E ) bar charts indicating the cell numbers of migrated or invasive cells per area. Magnification 200×. Scale bar: 100 μm. ( F , H ) Wound-healing assay of CAL27 cells incubated with Macro-CM from macrophages induced by TCM without or with rhENO1. ( F ) representative pictures; ( H ) bar charts indicating the wound healing rate. Magnification 40×. Scale bar: 500 μm. ( G,I,J ) Transwell assay for migration (upper panel) and invasion (lower panel) of CAL27 cells cocultured with macrophages induced by TCM without or with rhENO1. ( G ) representative pictures; ( I , J ) bar charts indicating the cell numbers of migrated or invasive cells per area. Magnification 200×. Scale bar: 100 μm. Different symbols (circle/square) were used to represent the data points of independent biological repeated experiments. All data are displayed as mean ± SEM; n = 3; ** p < 0.01 and *** p < 0.001.

Article Snippet: The levels of human ENO1 and murine IL-6 in the cell culture supernatants were measured by ELISA kits (Cusabio, Wuhan, China), following the manufacturer’s instructions.

Techniques: Migration, Wound Healing Assay, Incubation, Transfection, Transwell Assay

Journal: International Journal of Molecular Sciences

Article Title: ENO1 Promotes OSCC Migration and Invasion by Orchestrating IL-6 Secretion from Macrophages via a Positive Feedback Loop

doi: 10.3390/ijms24010737

Figure Lengend Snippet: ENO1 promotes epithelial–mesenchymal transition of tumor cells through macrophages. ( A ) Western blot representative image of E-cadherin, Vimentin and N-cadherin relative protein levels in CAL27 cells incubated with Macro-CM from macrophages induced by untreated (control), scrambled siRNA and ENO1-siRNA-transfected tumor supernatant, respectively, for 48 h. ( B ) Western blot representative image of E-cadherin, Vimentin and N-cadherin relative protein levels in CAL27 cells cocultured with Macro-CM from macrophage-induced tumor supernatant without or with rhENO1 for 48 h. ( C – H ) Statistical analysis of Western blot. Different symbols (circle/square/triangle) were used to represent the data points of independent biological repeated experiments. All data are displayed as mean ± SEM; n = 3; ns, no significance, * p < 0.05 and ** p < 0.01.

Article Snippet: The levels of human ENO1 and murine IL-6 in the cell culture supernatants were measured by ELISA kits (Cusabio, Wuhan, China), following the manufacturer’s instructions.

Techniques: Western Blot, Incubation, Control, Transfection

Journal: International Journal of Molecular Sciences

Article Title: ENO1 Promotes OSCC Migration and Invasion by Orchestrating IL-6 Secretion from Macrophages via a Positive Feedback Loop

doi: 10.3390/ijms24010737

Figure Lengend Snippet: ENO1 orchestrates IL-6 secretion of macrophages. ( A , B ) RT-qPCR analysis of the mRNA levels of cytokines ( Il6, Il10, Il12b, Tnfa and Tgfb1 ) in macrophages incubated with TCM from scrambled siRNA or ENO1-siRNA-transfected CAL27 for 6 h and 12 h, respectively. ( C , D ) RT-qPCR analysis of the mRNA levels of cytokines ( Il6, Il10, Il12b, Tnfa and Tgfb1 ) in macrophages incubated with TCM supplemented without or with rhENO1 for 6 h and 12 h, respectively. ( E ) ELISA for detection of IL-6 protein levels in CM harvested from macrophages incubated with TCM from scrambled siRNA or ENO1-siRNA-transfected CAL27 cells for 24 h. ( F ) ELISA for detection of IL-6 protein levels in CM harvested from macrophages incubated with TCM supplemented without or rhENO1 for 24 h. Different symbols (circle/square) were used to represent the data points of independent biological repeated experiments. All data are displayed as mean ± SEM; n = 3; * p < 0.05, ** p < 0.01 and **** p < 0.0001.

Article Snippet: The levels of human ENO1 and murine IL-6 in the cell culture supernatants were measured by ELISA kits (Cusabio, Wuhan, China), following the manufacturer’s instructions.

Techniques: Quantitative RT-PCR, Incubation, Transfection, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Molecular Sciences

Article Title: ENO1 Promotes OSCC Migration and Invasion by Orchestrating IL-6 Secretion from Macrophages via a Positive Feedback Loop

doi: 10.3390/ijms24010737

Figure Lengend Snippet: ENO1 orchestrates IL-6 secretion of macrophages via tumor cell-derived lactic acid. ( A , B ) CCK8 assay for cell viability of macrophages treated with indicated doses of lactic acid ( A ) or α-CHC ( B ) with TCM for 24 h. ( C , D ) RT-qPCR analysis of the mRNA levels of IL-6 in macrophages treated with 10 mM lactic acid ( C ) or 1mM α-CHC ( D ) with TCM for 24 h. ( E ) ELISA for detection of IL-6 protein levels in CM from macrophages treated with TCM without or with 10 mM lactic acid for 24 h. ( F ) ELISA for detection of IL-6 protein levels in CM from macrophages treated with TCM without or with 1 mM α-CHC for 24 h (1%DMSO in TCM was used as a solvent control). Different symbols (circle/square) were used to represent the data points of independent biological repeated experiments. All data are displayed as mean ± SEM; n = 3; * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001.

Article Snippet: The levels of human ENO1 and murine IL-6 in the cell culture supernatants were measured by ELISA kits (Cusabio, Wuhan, China), following the manufacturer’s instructions.

Techniques: Derivative Assay, CCK-8 Assay, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Solvent, Control

Journal: International Journal of Molecular Sciences

Article Title: ENO1 Promotes OSCC Migration and Invasion by Orchestrating IL-6 Secretion from Macrophages via a Positive Feedback Loop

doi: 10.3390/ijms24010737

Figure Lengend Snippet: ENO1 orchestrates IL-6 secretion of macrophages via paracrine ENO1/TLR4 signaling pathway. ( A ) Macrophages pretreated without or with TLR4 inhibitor (TAK242, 1 μM) supplemented in TCM were stimulated by rhENO1 for 12 h, and the mRNA levels of IL-6 in macrophages were detected by RT-qPCR analysis. ( B ) Macrophages pretreated without or with TLR4 inhibitor (TAK242, 1 μM) were stimulated by rhENO1 for 24 h, then IL-6 protein levels in CM were assayed by ELISA. ( C ) Immunofluorescence for colocalization of ENO1 and TLR4 on macrophages. Magnification 400×. Scale bar: 50μm. The area indicated on the upper left image was shown in a magnified image on the lower panel. Different symbols (circle/square/triangle) were used to represent the data points of independent biological repeated experiments. All data are displayed as mean ± SEM; n = 3; ** p < 0.01 and **** p < 0.0001.

Article Snippet: The levels of human ENO1 and murine IL-6 in the cell culture supernatants were measured by ELISA kits (Cusabio, Wuhan, China), following the manufacturer’s instructions.

Techniques: Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Immunofluorescence

Journal: International Journal of Molecular Sciences

Article Title: ENO1 Promotes OSCC Migration and Invasion by Orchestrating IL-6 Secretion from Macrophages via a Positive Feedback Loop

doi: 10.3390/ijms24010737

Figure Lengend Snippet: IL-6 promotes the migration and invasion of tumor cells. ( A ) Transwell assay for migration of CAL27 cells (upper chamber) pretreated without or with IL-6R antagonist tocilizumab (Toc, 5 μg/mL) and cocultured with macrophages (lower chamber) for 24 h. Left panel, representative pictures; right panel, bar charts indicating the cell numbers of migrated cells per area. Magnification 200×. Scale bar: 100 μm. ( B ) Transwell assay for invasion of CAL27 cells (upper chamber) pretreated without or with the IL-6R antagonist tocilizumab (Toc, 5 μg/mL) and cocultured with macrophages (lower chamber) for 48 h. Left panel, representative pictures; right panel, bar charts indicating the cell numbers of invasive cells per area. Magnification 200×. Scale bar: 100 μm. ( C ) Graphical abstract. ENO1 promotes tumor cell migration, invasion and EMT by orchestrating macrophage-derived IL-6 via secretion of lactic acid and extracellular ENO1 in OSCC, thus forming a positive feedback loop to promote OSCC progression. Different symbols (circle/square/triangle) were used to represent the data points of independent biological repeated experiments. All data are displayed as mean ± SEM; n = 3; * p < 0.05 and ** p < 0.01.

Article Snippet: The levels of human ENO1 and murine IL-6 in the cell culture supernatants were measured by ELISA kits (Cusabio, Wuhan, China), following the manufacturer’s instructions.

Techniques: Migration, Transwell Assay, Derivative Assay