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: ENO1 expression in liver cancer tissue and peripheral blood of patients with liver cancer. (A) ENO1 expression in liver cancer tissues (×100). (B) ENO1 was not expressed in benign liver lesions (×100). (C) Serum anti-ENO1 antibody levels in patients with liver cancer were higher than those in patients with benign liver lesions and healthy controls. (D) ENO1 expression in liver cancer tissues (×400). (E) ENO1 was not expressed in benign liver lesion (×400). (F) Receiver operating characteristic curve analysis for liver cancer diagnosis using anti-ENO1 antibody levels. The AUC was 0.741, the sensitivity was 64.3% and the specificity was 85.5%. The red dot represents the cut-off value. **P<0.01; ****P<0.0001 ENO1, α-enolase; AUC, area under the curve.

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

Techniques: Expressing

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: ENO1 expression in pathological tissues.

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

Techniques: Expressing

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:

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: Validation of the siRNA interference effect on ENO1 expression in liver cancer cells. (A) RT-qPCR validation of the effect of siRNA interference on ENO1 expression in HepG2 cells. The results showed that the relative expression levels of the ENO1 gene in the si-1 and si-2 groups were lower than those in the control group. (B) RT-qPCR validation of the effect of siRNA interference on ENO1 expression in Huh7 cells. The results showed that the relative expression levels of the ENO1 gene in the si-1 and si-2 groups were lower than those in the control group. (C) Western blot validation of the effect of siRNA interference on ENO1 expression in HepG2 cells. The results showed that the relative expression levels of the ENO1 protein in HepG2 cells in the si-1 and si-2 groups were significantly lower than those in the control group. (D) Western blot validation of the effect of siRNA interference on ENO1 expression in Huh7 cells. The results showed that the relative expression levels of the ENO1 protein in Huh7 cells in the si-1 and si-2 groups were significantly lower than those in the control group. **P<0.01; ****P<0.0001. siRNA, small interfering RNA; ENO1, α-enolase; si-1, siRNA interference group 1; si-2, siRNA interference group 2; RT-qPCR, reverse transcription-quantitative PCR.

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

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Small Interfering RNA, Real-time Polymerase Chain Reaction

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: Effect of ENO1 siRNA on liver cancer cell proliferation. (A) HepG2 and (B) Huh7 cell proliferation was suppressed after ENO1 siRNA treatment. Results show that 72 h after transfection in the si-1 and si-2 groups, ENO1 siRNA treatment resulted in proliferation inhibition in HepG2 and Huh7 cells. In HepG2 cells, the differences in cell proliferation between the NC and the siRNA-treated groups were statistically significant (P<0.05 vs. si-2 group and P<0.01 vs. si-1 group). Cell proliferation in Huh7 cells was also significantly different. *P<0.05; **P<0.01. ENO1, α-enolase; siRNA, small interfering RNA; si-1, siRNA interference group 1; si-2, siRNA interference group 2; OD, optical density; NC, negative control.

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

Techniques: Transfection, Inhibition, Small Interfering RNA, Negative Control

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: Effect of ENO1 siRNA on the migration ability of liver cancer cells. (A) Compared with that of the NC group, HepG2 cell migration after ENO1 siRNA treatment became slower in the si-1 and si-2 groups, and the difference was statistically significant at 48 h after treatment. (B) Gap size was measured using ImageJ software and the ratio of gap size after ENO1 siRNA treatment in HepG2 cells was calculated based on the size of the wound at 0 h. (C) Compared with that of the NC group, Huh7 cell migration after ENO1 siRNA treatment became slower in the si-1 and si-2 groups, and the difference was statistically significant at 48 h after treatment. (D) Gap size was measured with the ImageJ software and the ratio of gap size after ENO1 siRNA treatment in Huh7 cell was calculated based on the size of the wound at 0 h. *P<0.05; **P<0.01. ENO1, α-enolase; siRNA, small interfering RNA; NC, negative control; si-1, siRNA interference group 1; si-2, siRNA interference group 2.

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

Techniques: Migration, Software, Small Interfering RNA, Negative Control

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: Effect of ENO1 siRNA on the invasion and migration abilities of liver cancer cells. Compared with the NC group, the in vitro invasion and migration abilities of (A) HepG2 and (B) Huh7 cells after ENO1 siRNA treatment decreased in the si-1 and si-2 groups, and the differences were statistically significant. Numbers of (C) HepG2 and (D) Huh7 cells were measured with the ImageJ software after ENO1 siRNA treatment. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. ENO1, α-enolase; siRNA, small interfering RNA; NC, negative control; si-1, siRNA interference group 1; si-2, siRNA interference group 2.

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

Techniques: Migration, In Vitro, Software, Small Interfering RNA, Negative Control

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: 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: The antibodies used include rabbit polyclonal anti-ENO1 (OriGene) in conjunction with Alexa Fluor 488-anti-rabbit IgG (Invitrogen), APC-anti-CD44, PE-anti-CD90 (all from BD Biosciences), and/or PE-anti-CD133 (Biolegend, San Diego, CA).

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: 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: The antibodies used include rabbit polyclonal anti-ENO1 (OriGene) in conjunction with Alexa Fluor 488-anti-rabbit IgG (Invitrogen), APC-anti-CD44, PE-anti-CD90 (all from BD Biosciences), and/or PE-anti-CD133 (Biolegend, San Diego, CA).

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: sENO1 + CSCs generate more invadopodia than their sENO1 - counterparts. (A) Confocal views of sENO1 + PC-3 CSCs (represented by CD44 + CD133 + cells), sENO1 - CSCs, and non-CSCs (represented by cells in the other subpopulations) showing invadopodia (yellow puncta) with the colocalized cortactin (green) and F-actin (red) that penetrate the underlying gelatin matrix. Nuclei were counterstained with 4’,6-diamidino-2-phenylindole (DAPI; blue). Scale, 10 µm. (B) Quantification of the invadopodia density per cell in (A) . 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 < 0.001. (C) Representative immunoblots of ENO1, cortactin, and F-actin in the cell body (left) and the invadopodial (right) protein lysates fractionated from CD44 + CD133 + PC-3 CSCs seeded on gelatin for 6 hours. Protein levels were quantified by densitometric analysis of the bands, normalized to β-actin (loading control).

Article Snippet: The antibodies used include rabbit polyclonal anti-ENO1 (OriGene) in conjunction with Alexa Fluor 488-anti-rabbit IgG (Invitrogen), APC-anti-CD44, PE-anti-CD90 (all from BD Biosciences), and/or PE-anti-CD133 (Biolegend, San Diego, CA).

Techniques: Western Blot

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: 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 β-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; α-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 α-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 α-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 α-ENO1 or a control IgG (n = 50 cells counted per sample). Unpaired t-test was performed where ***p < 0.001.

Article Snippet: The antibodies used include rabbit polyclonal anti-ENO1 (OriGene) in conjunction with Alexa Fluor 488-anti-rabbit IgG (Invitrogen), APC-anti-CD44, PE-anti-CD90 (all from BD Biosciences), and/or PE-anti-CD133 (Biolegend, San Diego, CA).

Techniques: Western Blot, shRNA, Expressing, Fluorescence, Staining, Invasion Assay, Immunofluorescence, Concentration Assay, Immunostaining

Journal: Journal of Gastrointestinal Oncology

Article Title: RNA-binding protein ENO1 promotes the tumor progression of gastric cancer by binding to and regulating gastric cancer-related genes

doi: 10.21037/jgo-23-151

Figure Lengend Snippet: Characterization of the ENO1 -RNA interaction profile by an iRIP-seq analysis. (A) Experimental and computational work flow of the iRIP-seq. (B) Western blot analysis of the ENO1 immunoprecipitates using anti-Flag monoclonal antibody. Two replicates were performed. (C) Sample cluster analysis plots. (D) Sample correlation analysis. (E) A pie chart showing the genomic distribution of the ENO1 -bound peaks from the two biological replicates. (F) The motif analysis results showing the enriched motifs from the ENO1 -bound peaks from the two biological replicates. (G) The distribution of the GACGAGGA enrichment sequences in both experiments in the Ablife analysis. (H) A Venn diagram showing the overlapping peaks between IP-1 and IP-2. (I) A scatter plot showing the most enriched GO biological process (left panel) and KEGG pathway (right panel) results of the overlapping peak genes. ENO1 , enolase 1; IgG, immunoglobulin G; RT-PCR, reverse transcription-polymerase chain reaction; IP, immunoprecipitation; PPIA, peptidylprolyl isomerase A; 3'UTR, 3' untranslated region; 5'UTR, 5' untranslated region; CDS, coding sequence; nc, non-coding; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; iRIP-seq, improved RNA immunoprecipitation and deep sequencing.

Article Snippet: ENO1 antibody was purchased from Abcam (Cambridge, UK).

Techniques: Western Blot, Reverse Transcription Polymerase Chain Reaction, Immunoprecipitation, Sequencing

Journal: Journal of Gastrointestinal Oncology

Article Title: RNA-binding protein ENO1 promotes the tumor progression of gastric cancer by binding to and regulating gastric cancer-related genes

doi: 10.21037/jgo-23-151

Figure Lengend Snippet: ENO1 binds to genes related to the occurrence and development of GC. (A) The ENO1 -binding peak genes of NEAT1 . Left panel: an IGV-sashimi plot showing the peak reads and binding sites across the mRNA; the green and red panels represent the positions of the peaks. The distribution reads of the bound gene are plotted in the upper panel and the transcripts of each gene are shown below. Right panel: a bar plot showing the FPKM of NEAT1 . (B) The ENO1 -binding peak genes of PKM . Left panel: an IGV-sashimi plot showing the peak reads and binding sites across the mRNA; the green and blue panels represent the positions of the peaks. The distribution reads of the bound gene is plotted in the upper panel and the transcripts of each gene are shown below. Right panel: a bar plot showing the FPKM of PKM . (C) The ENO1 -binding peak genes of CD44 . Left panel: an IGV-sashimi plot showing the peak reads and binding sites across the mRNA; the green and red panels represent the positions of the peaks. The distribution reads of the bound gene is plotted in the upper panel, and the transcripts of each gene are shown below. Right panel: a bar plot showing the FPKM of CD44 . **, P<0.05; ***, P<0.001. ENO1, enolase 1; IP, immunoprecipitation; NEAT1, nuclear enriched abundant transcript 1; PKM, pyruvate kinase M; GC, gastric cancer; IGV, Integrative Genomics Viewer; FPKM, fragments per kilobase million.

Article Snippet: ENO1 antibody was purchased from Abcam (Cambridge, UK).

Techniques: Binding Assay, Immunoprecipitation

Journal: Journal of Gastrointestinal Oncology

Article Title: RNA-binding protein ENO1 promotes the tumor progression of gastric cancer by binding to and regulating gastric cancer-related genes

doi: 10.21037/jgo-23-151

Figure Lengend Snippet: The ENO1 -interaction regulates the expression of the target genes associated with GC. The differentially expressed genes were output by using the data of previous gene chip sequencing. (A) Left panel: a Venn diagram showing the overlapping genes between the ENO1 -bound peaks in the fRIP-seq and the upregulated DEGs. Right panel: the overlapping genes between the ENO1 -bound peaks in the fRIP-seq and the downregulated DEGs. (B) A bar plot showing the expression pattern and statistical differences of the upregulated DEGs in the overlapping genes. The error bars represent the mean ± SEM. (C) A bar plot showing the expression pattern and statistical difference of the downregulated DEGs in the overlapping genes. The error bars represent the mean ± SEM. (D) The ENO1 -binding peak genes of MCL1 . An IGV-sashimi plot showing the peak reads and binding sites across the mRNA; the green and blue panels represent the positions of the peaks. The read distribution of the bound gene is plotted in the upper panel and the transcripts of each gene are shown below. (E) The ENO1 -binding peak genes of SOX9 . An IGV-sashimi plot showing the peak reads and binding sites across the mRNA; the green and blue panels represent the positions of the peaks. The read distribution of the bound gene is plotted in the upper panel and the transcripts of each gene are shown above. ***, P<0.001. DEG, differentially expressed gene; IP, immunoprecipitation; NC, negative control; KD, knockdown; MCL1, myeloid cell leukemia 1; VEGFA, vascular endothelial growth factor A; SOX9, SRY-box transcription factor 9; ENO1, enolase 1; GC, gastric cancer; fRIP-seq, formaldehyde crosslinking RNA immunoprecipitation sequencing; SEM, standard error of the mean; IGV, Integrative Genomics Viewer.

Article Snippet: ENO1 antibody was purchased from Abcam (Cambridge, UK).

Techniques: Expressing, ChIP-sequencing, Binding Assay, Immunoprecipitation, Negative Control, Sequencing