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 FASEB Journal

Article Title: PHD3 is a transcriptional coactivator of HIF-1α in nucleus pulposus cells independent of the PKM2-JMJD5 axis

doi: 10.1096/fj.201601291R

Figure Lengend Snippet: PHD3−/− mice show increased incidence of disc degeneration and decreased expression of select HIF-1 target genes in NP tissue. A) Schematic drawing of spinal motion segment and coronal cross section showing vertebral bodies and the intervertebral disc with its central NP, circumferential AF, and superior and inferior cartilaginous endplates (CEP). B, C) Representative Safranin-O/Fast Green/hematoxylin–stained sections of 12.5-mo-old WT (B) and PHD3−/− (C) mice. The PHD3−/− mouse showed a decreased number of NP cells and some changes in cell morphology. D) PHD3−/− mice showed increased incidence of discs with a higher grade of degeneration, as assessed by the Thompson grading scale. Four discs/animal were scored from 3 pairs of littermate animals. E–L') Representative immunofluorescence images from 12.5-mo-old WT (PHD3+/+) and knockout (PHD3−/−) mice showing NP tissue areas with a comparable number of cells. There was decreased staining of HIF-1 targets VEGF-A (E–F'), LDHA (G–H'), and GLUT1 (I, J) in knockout mice compared to WT mice. In contrast, another HIF-1 target, ENO1, shows comparable expression in PHD3+/+ (K, K') and PHD3−/− (L, L') mice. Krt19 was used as a marker to label the NP tissue compartment with VEGF-A, LDHA, and ENO1 (independent channel not shown). Scale bars, 100 μm (B, C) and 50 μm (E–L'). Images are representative from 3 independent littermate groups.

Article Snippet: Sections were then sequentially incubated with antibodies [VEGF, ab46154; LDHA, ab52488; and GLUT1, ab40084 (all from Abcam), or ENO1, NB100-65252 (Novus Biologics, Littleton CO, USA), all at a concentration of 1:100) with an NP marker anti-keratin (Krt)19 antibody (1:3, TROMA-III; Developmental Studies Hybridoma Bank, Iowa City, IA, USA) in 5% normal donkey serum in 1× PBS, 0.4% Triton-X at 4°C overnight ( 22 ).

Techniques: Expressing, Staining, Immunofluorescence, Knock-Out, Marker

Journal: The FASEB Journal

Article Title: PHD3 is a transcriptional coactivator of HIF-1α in nucleus pulposus cells independent of the PKM2-JMJD5 axis

doi: 10.1096/fj.201601291R

Figure Lengend Snippet: PHD3 controls expression of a select set of HIF-1 targets in NP cells through a p300-dependent mechanism. A, B) Bright-field (A) and fluorescent (B) images of rat NP cells after transduction with lentivirus coexpressing shPHD3 and enhanced green fluorescent protein. Scale bars, 10 μm. C) Measurement of PHD3 mRNA expression after transduction of NP cells with shRNA targeting PHD3 (n = 7). D) Measurement of mRNA expression of HIF-1 target genes in PHD3-silenced NP cells cultured in NX or HX for 72 h (n = 7). E) Hypoxic expression of Pgk1, Gapdh, and Eno1 in PHD3-silenced cells did not change (n = 3). F) Measurement of activity of Eno1 promoter containing 2 well-characterized HIF-1 binding sites after PHD3 knockdown (n = 7). G, H) Western blot (G) and corresponding densitometric analysis (H) of select HIF-1 targets in NP cells after stable knockdown of PHD3 (n = 4). I) Location of HRE sites within promoters of Vegfa, Slc2a1, and Ldha and location of ChIP primers used in J. J) HIF-1α enrichment at HRE sites within Vegfa, Slc2a1, and Ldha promoters decreases after knockdown of PHD3 during 72 h HX (n = 3). Luciferase assays were performed with 3 technical replicates per experiment; quantitative RT-PCR assays were performed with 2 technical replicates per experiment. Data are means ± sem. *P < 0.05.

Article Snippet: Sections were then sequentially incubated with antibodies [VEGF, ab46154; LDHA, ab52488; and GLUT1, ab40084 (all from Abcam), or ENO1, NB100-65252 (Novus Biologics, Littleton CO, USA), all at a concentration of 1:100) with an NP marker anti-keratin (Krt)19 antibody (1:3, TROMA-III; Developmental Studies Hybridoma Bank, Iowa City, IA, USA) in 5% normal donkey serum in 1× PBS, 0.4% Triton-X at 4°C overnight ( 22 ).

Techniques: Expressing, Transduction, shRNA, Cell Culture, Activity Assay, Binding Assay, Knockdown, Western Blot, Luciferase, Quantitative RT-PCR

Journal: MedComm

Article Title: Global Lactylome Reveals Lactylation‐Dependent Mechanisms Underlying CXC Motif Chemokine Ligand 12 Expression in Pulmonary Endothelium During Acute Respiratory Distress Syndrome

doi: 10.1002/mco2.70344

Figure Lengend Snippet: Increased protein lactylation in the PECs of ARDS mice, with Eno1 as a substrate. (A) Histogram of the ratio distribution of quantifiable Klac sites in the PECs of control and LPS‐induced ARDS mice. (B) GO items associated with Klac sites (ARDS/Control > +∞) located proteins. (C) Venn diagram of predicted CXCL12 3′ UTR binding proteins and proteins in picked GO items and the average protein copies of 13 screened proteins in PECs. (D) Ribbon diagram of the crystal structure of mouse Eno1 and lactylation at the K193 residue. (E) Mass spectrometry of Eno1 lactylated at K193. (F) Purified mouse Eno1 (100 µg/mL) was incubated with different concentrations of lactyl‐CoA for 1 h at 37°C. Then, the mixtures were added to protein loading buffer for denaturation, and K‐lactylation was assessed by western blotting. (G) The K193 site in Eno1 is conserved in different species. Conserved K193 sites are marked in red in the sequences among different species. (H) Western blotting analysis of the K193 lactylated level in PECs isolated from the lungs of control and LPS‐induced ARDS mice. (I) In vitro, the K193 lactylation levels in PBS‐ and LPS‐activated MPMECs were measured by western blotting.

Article Snippet: The following primary antibodies were used: anti‐L‐lactyl lysine (PTM‐1401, PTM Biolabs, China), anti‐mouse Eno1 (A1033, ABclonal, China), anti‐K193 lactylated Eno1 (custom designed, ABclonal, China), and anti‐mouse β‐actin (ZF0033, ZFdows Bio, China).

Techniques: Control, Binding Assay, Residue, Mass Spectrometry, Purification, Incubation, Western Blot, Isolation, In Vitro

Journal: MedComm

Article Title: Global Lactylome Reveals Lactylation‐Dependent Mechanisms Underlying CXC Motif Chemokine Ligand 12 Expression in Pulmonary Endothelium During Acute Respiratory Distress Syndrome

doi: 10.1002/mco2.70344

Figure Lengend Snippet: K193 lactylated Eno1 released the bound CXCL12 mRNA to accelerate translation. (A) Eno1 expression in MPMECs transfected with siRNAs. (B) CXCL12 mRNA and (C) protein expression in Eno1 KD cells after LPS stimulation ( n = 3). (D) Molecular docking and ΔG binding energy calculation (E) RIP‐qPCR detection of Eno1‐bound CXCL12 transcripts in MPMECs treated with LPS ( n = 3). (F) Click‐iT AHA assays to detect the CXCL12 translation efficacy in MPMECs treated with LPS ( n = 3). (G) Schematics of the reporter and effector constructs. Dual‐luciferase analysis in HEK293T cells transfected with the reporter expressing the CXCL12‐3′ UTR and constructs expressing the WT, K193Q, or K193R Eno1 ( n = 3). (H) CXCL12 concentrations in culture media from MPMECs transfected with shEno1, shEno1+oe‐WT, or shEno1+oe‐K193R ( n = 3). * p < 0.05, ** p < 0.01, **** p < 0.001.

Article Snippet: The following primary antibodies were used: anti‐L‐lactyl lysine (PTM‐1401, PTM Biolabs, China), anti‐mouse Eno1 (A1033, ABclonal, China), anti‐K193 lactylated Eno1 (custom designed, ABclonal, China), and anti‐mouse β‐actin (ZF0033, ZFdows Bio, China).

Techniques: Expressing, Transfection, Binding Assay, Construct, Luciferase

Journal: MedComm

Article Title: Global Lactylome Reveals Lactylation‐Dependent Mechanisms Underlying CXC Motif Chemokine Ligand 12 Expression in Pulmonary Endothelium During Acute Respiratory Distress Syndrome

doi: 10.1002/mco2.70344

Figure Lengend Snippet: K193 lactylation augmented Eno1 enzymatic activity. (A) The levels of lactate in the culture medium of PBS‐ and LPS‐treated MPMECs over time ( n = 3). (B) PBS‐ and LPS‐treated MPMECs lysates were prepared to measure Eno1 enzymatic activity by monitoring phosphoenolpyruvate concentrations over time ( n = 3). (C) Eno1 enzymatic activity was measured in lysates from shEno1+oe‐WT and shEno1+oe‐K193R MPMEC ( n = 6). * p < 0.05. In C, *compared with the untreated shEno1+oe‐WT group, * p < 0.05; # compared with the lactate‐treated shEno1+oe‐K193R group, # p < 0.05.

Article Snippet: The following primary antibodies were used: anti‐L‐lactyl lysine (PTM‐1401, PTM Biolabs, China), anti‐mouse Eno1 (A1033, ABclonal, China), anti‐K193 lactylated Eno1 (custom designed, ABclonal, China), and anti‐mouse β‐actin (ZF0033, ZFdows Bio, China).

Techniques: Activity Assay

Journal: Biochemistry and biophysics reports

Article Title: Icariin promotes osteogenic differentiation by upregulating alpha-enolase expression.

doi: 10.1016/j.bbrep.2023.101471

Figure Lengend Snippet: Fig. 2. Eno1 was upregulated in icariin-induced osteogenic differentiation. (A) Venn analysis of targets of postmenopausal osteoporosis (PMOP) and diabetes mellitus (DM). (B) KEGG pathway enrichment analysis was performed. Red box indicates HIF-1 signalling pathway. (C) Protein-protein interaction network was built and Eno1 was marked in red. (D)mRNA levels of Eno1 was tested by qPCR. All the data are shown as mean ± SD (n = 3) in three independent experiments. (E) Western Blot analysis for Eno1 was performed. Vinculin was used as a reference protein. Relative expression of Eno1(F) were normalized against Vinculin. *P＜0.05, ***P＜0.001 versus the blank control group. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: To determine the role of Eno1 in osteogenic differentiation and icariin efficacy, osteogenic differentiation culture medium containing 5 μM ENOblock (MedChemExpress) was used.

Techniques: Western Blot, Expressing, Control

Journal: Biochemistry and biophysics reports

Article Title: Icariin promotes osteogenic differentiation by upregulating alpha-enolase expression.

doi: 10.1016/j.bbrep.2023.101471

Figure Lengend Snippet: Fig. 3. Inhibition of Eno1 weakened icariin-induced osteogenic differentiation. (A) Cell viability of MC3T3-E1 under different concentration of ENOblock were measured by CCK8 assay for 24, 48, 72h. (B) The mineralization of 21-days induction with or without ENOblock and icariin were performed by Alizarin red S staining, and calcium deposits were quantified by measuring OD562 after destaining (C). (D–F) mRNA levels of Alp (D), Bgp (E) and Runx2 (F), were tested as osteogenic markers by qPCR. All the data are shown as mean ± SD (n = 3) in three independent experiments. *P＜0.05, **P＜0.01, ****P＜0.0001 versus the blank control group, &&P＜0.01, &&&&P＜0.0001 versus the icariin group. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: To determine the role of Eno1 in osteogenic differentiation and icariin efficacy, osteogenic differentiation culture medium containing 5 μM ENOblock (MedChemExpress) was used.

Techniques: Inhibition, Concentration Assay, CCK-8 Assay, Staining, Control

Journal: Biochemistry and biophysics reports

Article Title: Icariin promotes osteogenic differentiation by upregulating alpha-enolase expression.

doi: 10.1016/j.bbrep.2023.101471

Figure Lengend Snippet: Fig. 4. BMP/Smad4 signalling pathway altered in Eno1-regulated osteogenic differentiation. (A) Western Blot analysis for BMP4, BMP2, Smad4, p-Smad1/5/9 and Smad1/5/9 were performed. Vinculin was used as a reference protein. Relative expression of BMP4 (B), BMP2 (C) and Smad4 (D) were normalized against Vinculin. (E)The ratio of p-Smad1/5/9 and Smad1/5/9 was displayed. All the data are shown as mean ± SD (n = 3) in three independent experiments. *P＜0.05, **P ＜0.01versus the blank control group, &&P＜0.01, &&&P＜0.001 versus the icariin group.

Article Snippet: To determine the role of Eno1 in osteogenic differentiation and icariin efficacy, osteogenic differentiation culture medium containing 5 μM ENOblock (MedChemExpress) was used.

Techniques: Western Blot, Expressing, Control

Journal: EBioMedicine

Article Title: Anti α-enolase antibody is a novel autoimmune biomarker for unexplained recurrent miscarriages

doi: 10.1016/j.ebiom.2019.02.027

Figure Lengend Snippet: Anti-ENO1 antibodies inhibit the expression of β-hCG and progesterone in JEG-3 cells. (a) An obvious band signal marked by an arrow was detected by 1 dimension western blot. JEG-3 proteins were used as antigens, 10 μg/ml ATAB-positive IgGs as the primary antibody and goat-anti-human antibodies as the secondary antibody. ENO1 was identified to be one of the most prominent targets with western blot and mass spectrometry. 1D western blots were repeated at least 5 times. (b.c) The production of β-hCG in JEG-3 cells was inhibited by 1, 100 and 1000 ng/ml of anti-ENO1 antibodies while the secretion of progesterone was suppressed by 1 μg/ml of anti-ENO1 antibodies compared to the rabbit polyclonal IgGs by ELISA (P < 0·05, Wilcoxon test, n = 6). Data were shown as mean ± SEM and P-values are shown individual treatment groups compared to isotype control.

Article Snippet: On the basis of 1D western blotting, a competition assay was performed with similar procedures except for the primary antibodies, where a mixture of ATAB-positive IgGs and polyclonal rabbit anti-ENO1 antibodies (Novus Biologicals, Littleton, Colorado, USA) both at a concentration of 10 μg/ml was used.

Techniques: Expressing, Western Blot, Mass Spectrometry, Enzyme-linked Immunosorbent Assay, Control

Journal: EBioMedicine

Article Title: Anti α-enolase antibody is a novel autoimmune biomarker for unexplained recurrent miscarriages

doi: 10.1016/j.ebiom.2019.02.027

Figure Lengend Snippet: Anti-ENO1 antibodies are a member of ATAB. (a) The signals of ENO1 in JEG-3 cells were reduced by incubation with a mixture of antibodies (10 μg/ml rabbit anti-ENO1 antibodies and 10 μg/ml human ATAB-positive IgGs) compared with only 10 μg/ml incubating human purified IgGs or 10 μg/ml rabbit anti-ENO1 antibodies. Different secondary antibodies were used in western blots: goat-anti-human antibody, goat-anti-human antibody and goat-anti-rabbit antibody (from left to right rows). (b.c) The diagram shows that in the flow cytometry mixed antibodies (0·42 mg/ml anti-ENO1 antibodies with 2 mg/ml isolated IgGs) bind less JEG-3 cells as ATAB-positive IgGs (P < 0·05, Wilcoxon test, n = 3). (d.e) Pre-incubating 20 μg recombinant human ENO1 protein with 2 mg/ml ATAB-positive IgGs decreases the binding of ATAB-positive IgGs to JEG-3 cells (P < 0·05, Wilcoxon test, n = 3). The MCS values are showed with mean ± SD and P-values are shown individual treatment groups compared to incubation with ATAB-positive IgGs.

Article Snippet: On the basis of 1D western blotting, a competition assay was performed with similar procedures except for the primary antibodies, where a mixture of ATAB-positive IgGs and polyclonal rabbit anti-ENO1 antibodies (Novus Biologicals, Littleton, Colorado, USA) both at a concentration of 10 μg/ml was used.

Techniques: Incubation, Purification, Western Blot, Flow Cytometry, Isolation, Recombinant, Binding Assay

Journal: EBioMedicine

Article Title: Anti α-enolase antibody is a novel autoimmune biomarker for unexplained recurrent miscarriages

doi: 10.1016/j.ebiom.2019.02.027

Figure Lengend Snippet: Anti-ENO1 antibodies inhibit the expression of β-hCG and progesterone in human villous trophoblast (HVT) cells. (a) The production of β-hCG in HVT cells was inhibited by 1, 10 and 100 ng/ml of anti-ENO1 antibodies in a dose dependent manner. (each P value <0·05, Wilcoxon test, n = 6). (b) The secretion of progesterone in HVT cells was suppressed by 1, 10 and 100 ng/ml of anti-ENO1 antibodies in a dose dependant manner. (each P value <0·05, Wilcoxon test, n = 6). Values are expressed as shown as mean ± SEM and P-values are shown individual treatment groups compared to isotype control.

Article Snippet: On the basis of 1D western blotting, a competition assay was performed with similar procedures except for the primary antibodies, where a mixture of ATAB-positive IgGs and polyclonal rabbit anti-ENO1 antibodies (Novus Biologicals, Littleton, Colorado, USA) both at a concentration of 10 μg/ml was used.

Techniques: Expressing, Control

Journal: EBioMedicine

Article Title: Anti α-enolase antibody is a novel autoimmune biomarker for unexplained recurrent miscarriages

doi: 10.1016/j.ebiom.2019.02.027

Figure Lengend Snippet: Anti-ENO1 titers are higher in the sera of ATAB-positive patients. (a) Anti-ENO1 antibodies were increased in the 16 uRM patients with positive ATAB compared with 17 cases with negative ATAB (P = 0·007, student's t -test). (b.c) The levels of anti-γ-Enolase antibodies and anti-citrullinated fibrinogen (CFG) antibodies were not changed in 36 ATAB-positive patients and 30 ATAB-negative cases (both P > 0·05, student's t -test). (d) Anti-MCV antibodies were higher in the sera of 34 ATAB-positive patients than 28 ATAB-negative cases (P = 0·018, student's t -test). Data were shown as mean ± SEM and statistically significant differences between two uRM groups were marked with P values.

Article Snippet: On the basis of 1D western blotting, a competition assay was performed with similar procedures except for the primary antibodies, where a mixture of ATAB-positive IgGs and polyclonal rabbit anti-ENO1 antibodies (Novus Biologicals, Littleton, Colorado, USA) both at a concentration of 10 μg/ml was used.

Techniques:

Journal: EBioMedicine

Article Title: Anti α-enolase antibody is a novel autoimmune biomarker for unexplained recurrent miscarriages

doi: 10.1016/j.ebiom.2019.02.027

Figure Lengend Snippet: ENO1 expression is increased in the decidua of uRM patients in the first trimester. Immunohistological analyses of ENO1 expression in the first trimester placenta were measured among 20 uRM patients and 22 healthy controls by IRS scores. (a–c) In the syncytium, the staining of ENO1 did not show significant difference in uRM patients (a) and controls (b), which is represented as bar charts (P > 0·05, Mann-Whitney test). The strong staining of ENO1 in the syncytium is marked with red arrows. (d–f) In the decidua, the expression of ENO1 was higher in uRM patients (d) as in controls (e, P = 0·038, Mann-Whitney test). Magnification × 10 lens, scale bar = 200 μm; magnifications of inserted pictures are ×25 lens, scale bar = 100 μm. uRM = unexplained recurrent miscarriages. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: On the basis of 1D western blotting, a competition assay was performed with similar procedures except for the primary antibodies, where a mixture of ATAB-positive IgGs and polyclonal rabbit anti-ENO1 antibodies (Novus Biologicals, Littleton, Colorado, USA) both at a concentration of 10 μg/ml was used.

Techniques: Expressing, Staining, MANN-WHITNEY

Journal: EBioMedicine

Article Title: Anti α-enolase antibody is a novel autoimmune biomarker for unexplained recurrent miscarriages

doi: 10.1016/j.ebiom.2019.02.027

Figure Lengend Snippet: ENO1 coexpresses with β-arrestin in the extravillous trophoblasts (EVT) of uRM patients and healthy controls. Confocal microscopy images showed a co-localization of ENO1 (red) and β-arrestin (green) in EVT of the first trimester placenta in uRM women (a–d) and healthy controls (e–h), which are shown with white arrows. HLA-G is used as the marker for EVT (magenta), magnification x20, scale bar =20 μM. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: On the basis of 1D western blotting, a competition assay was performed with similar procedures except for the primary antibodies, where a mixture of ATAB-positive IgGs and polyclonal rabbit anti-ENO1 antibodies (Novus Biologicals, Littleton, Colorado, USA) both at a concentration of 10 μg/ml was used.

Techniques: Confocal Microscopy, Marker

Journal: EBioMedicine

Article Title: Anti α-enolase antibody is a novel autoimmune biomarker for unexplained recurrent miscarriages

doi: 10.1016/j.ebiom.2019.02.027

Figure Lengend Snippet: The possible role of ENO1 in unexplained recurrent miscarriages (uRM). Anti-ENO1 antibodies are among the antitrophoblast antibodies (ATAB) and are higher expressed in the sera of positive-ATAB uRM patients. The translocation (1), overexpression (2) or posttranslational modifications (3) of ENO1 in the extravillous trophoblasts may be the reason for induction of anti-ENO1 antibody expression in the sera of uRM patients. Anti-ENO1 antibodies can inhibit the production of β-hCG and progesterone in trophoblast cells, and may stimulate the expression of PAI-1. All these changes may eventually contribute to recurrent miscarriages.

Article Snippet: On the basis of 1D western blotting, a competition assay was performed with similar procedures except for the primary antibodies, where a mixture of ATAB-positive IgGs and polyclonal rabbit anti-ENO1 antibodies (Novus Biologicals, Littleton, Colorado, USA) both at a concentration of 10 μg/ml was used.

Techniques: Translocation Assay, Over Expression, Expressing