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: Advanced Science

Article Title: Granzyme B PET Imaging Reveals Lgmn + Macrophage‐Mediated Immune Evasion and Guides Immunotherapy in EGFR‐TKI‐Resistant NSCLC

doi: 10.1002/advs.77837

Figure Lengend Snippet: Lgmn recruits Chip to ubiquitinate Eno1 to regulate the dysfunction of CD8 + T cells. (A) The expression of glycolysis‐related enzymes in CD8 + T cells treated with Lgmn fl/fl /EVs and Lgmn cko /EVs from proteomics analysis by LC‐MS/MS. (B) Cell lysates were immunoprecipitated using anti‐Lgmn antibodies, and Eno1 expression was detected by Western blotting. (C, D) Eno1 expression in CD8 + T cells treated with Lgmn fl/fl /EVs and Lgmn cko /EVs was detected by qRT‐PCR (C) and Western blotting (D). (E, F) Detection of Eno1 expression in CD8 + T cells overexpressing Lgmn by immunoblotting (E) and qRT‐PCR (F). (G, H) Detection of Eno1 protein stability in CD8 + T cells overexpressing Lgmn (G) or treated with EVs (H) by immunoblotting. (I) Detect Eno1 expression in CD8 + T cells overexpressing Lgmn or treated with MG132 using Western blotting. (J) Detect Eno1 expression in CD8 + T cells overexpressing Lgmn or treated with CQ using Western blotting. (K) Detect Eno1 ubiquitination in CD8 + T cells overexpressing Lgmn using Western blotting. (L) Cell lysates were immunoprecipitated using anti‐Lgmn or anti‐Eno1 antibodies, and the proteins interacting with Lgmn and Eno1 were detected by LC‐MS. (M) Co‐IP assays were conducted to observe the interaction between Lgmn, Chip, and Eno1. (N) Chip was knocked down, and Eno1 ubiquitination in CD8 + T cells was detected by Western blotting. (O) Chip was knocked down, and Lgmn was overexpressed; Eno1 expression and ubiquitination in CD8 + T cells were detected by Western blotting. (P) Cell lysates were immunoprecipitated using anti‐Eno1 antibodies, and Chip expression was detected by Western blotting. (Q, R) Chip was knocked down, and Lgmn was overexpressed; glucose uptake in CD8 + T cells was detected (Q), and TIM‐3 + and PD‐1 + CD8 + T cells were detected by flow cytometry (R). (S) Illustration of Lgmn enhancing the interaction between Chip and Eno1, and subsequently modulating CD8 + T cell exhaustion through metabolic reprogramming.

Article Snippet: Primary antibodies used are as follows: anti‐β‐actin (#4970, Cell Signaling Technology, 1:2000), anti‐Lgmn (A23776, ABclonal, 1:1000), anti‐Eno1 (A11448, ABclonal, 1:1000), anti‐Chip (#2080, Cell Signaling Technology, 1:1000), anti‐HA (AE105, ABclonal, 1:20000), anti‐FLAG (AE005, ABclonal, 1:10000), anti‐Ubiquitin (A19686, ABclonal, 1:1000).

Techniques: Expressing, Liquid Chromatography with Mass Spectroscopy, Immunoprecipitation, Western Blot, Quantitative RT-PCR, Ubiquitin Proteomics, Co-Immunoprecipitation Assay, Flow Cytometry

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: 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: 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