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: American Journal of Translational Research

Article Title: Reduced expression of enolase-1 correlates with high intracellular glucose levels and increased senescence in cisplatin-resistant ovarian cancer cells

doi:

Figure Lengend Snippet: ENO1 protein and mRNA levels in a panel of ovarian cancer cells. (A) Western blot analysis was performed using protein extracts (30-50 µg) of cisplatin-sensitive (A2780, OV-90, and OVCAR3) and cisplatin-resistant (A280CP20, A2780CIS, OV-90CIS, and OVCAR3CIS) ovarian cancer cells. (B) Densitometry analysis of band intensities, shown in (A). Fold changes in protein levels were calculated relative to the cisplatin sensitive pair. Averages ± SEM are shown for three independent experiments (C) ENO1 mRNA expression levels were assessed by qPCR. β-actin was used as a PCR internal control. Fold changes in mRNA levels were calculated relative to the cisplatin sensitive pair. Averages ± SEM are shown for three independent experiments. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

Article Snippet: The next day, 2 µg of ENO1 or Empty Vector in combination with MegaTran 1.0 Transfection reagent (ratio 1:1.5 w/v) (OriGene, catalog # TT200002) was added to each well.

Techniques: Western Blot, Expressing

Journal: American Journal of Translational Research

Article Title: Reduced expression of enolase-1 correlates with high intracellular glucose levels and increased senescence in cisplatin-resistant ovarian cancer cells

doi:

Figure Lengend Snippet: In vitro effect of ENO1 overexpression or silencing on cell growth and proliferation. (A) A2780CP20 cells were stably transfected with an empty vector (EV) or with an ENO1-containing vector. Western blot analysis was performed with 50 µg of protein extracts. (B) EV and ENO1 clones (3 × 104 cell/ml) were exposed to different concentrations of cisplatin for 72 h. Cell viability values were calculated relative to untreated cells. (C, D) Percentages of clonogenicity were calculated relative to EV cells. (E) A2780 cells (3 × 104 cells/ml) were transiently transfected with a negative control siRNA (NC-siRNA) or the two ENO1-targeted siRNAs. Western blot was performed with 50 µg of protein extracts. (F) Densitometry analysis of band intensities from (E). Protein levels were calculated relative to NT cells. (G) A2780 cells (3 × 104 cells/ml) cells were plated in 96-wells and the next day cells were transfected with different concentrations of siRNAs, as described in (E) Twenty-four hours after siRNA transfection, cisplatin (2 mM, final concentration) was added to the cells. Forty-eight hours later, cell viability was measured. Averages ± SEM are shown for three independent experiments. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

Article Snippet: The next day, 2 µg of ENO1 or Empty Vector in combination with MegaTran 1.0 Transfection reagent (ratio 1:1.5 w/v) (OriGene, catalog # TT200002) was added to each well.

Techniques: In Vitro, Over Expression, Stable Transfection, Transfection, Plasmid Preparation, Western Blot, Clone Assay, Negative Control, Concentration Assay

Journal: Cancer cell

Article Title: Integrative plasma and fecal metabolomics identify functional metabolites in adenoma-colorectal cancer progression and as early diagnostic biomarkers.

doi: 10.1016/j.ccell.2024.07.005

Figure Lengend Snippet: Figure 6. Identification of binding receptors of key metabolites in CRC (A) Experimental schematic of LiP-MS and volcano plot of differential peptides in CRC cells after oleic acid treatment. (B and C) Molecular docking analysis (B) and SPR assay (C) of the binding between oleic acid and ENO1. (D and E) Cell viability at OD570 of oleic acid-treated CRC cells after silencing ENO1 (D) or using ENO1 antagonist AP-III-a4 (E). (F) Experimental schematic and volcano plot of differential peptides in CRC cells after allocholic acid treatment. (G and H) Molecular docking analysis (G) and SPR assay (H) of the binding between allocholic acid and FXR1. (I and J) Cell viability at OD570 of allocholic acid-treated CRC cells after silencing FXR1 (I) or using FXR1 antagonist glycine-b-muricholic acid (J). (K and L) Colony formation of allocholic acid-treated CRC cells after silencing FXR1 (K) or using FXR1 antagonist (L). (M) Tumor parameters of CRC xenograft mice bearing HCT116 tumors (n = 6 per group). AA, allocholic acid; CA, cholic acid; Gly-b-MCA, glycine-b-muricholic acid; KD, dissociation constant; Scr, scrambled. Results are shown as mean ± SD. Each spot represents one sample or replicate. Experiment was conducted with triplicates (A–C, F–H, K, and L) or 5 replicates (D, E, I, and J) per group. Wilcoxon rank-sum test (A and F), repeated measures two-way ANOVA (D, E, I, J, and M/top), or Mann-Whitney U test (K, L, and M/bottom) was used to examine the statistical significance. See also Figure S6 and Table S4.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER CytoBuster Protein Extraction Reagent Merck Millipore Cat#71009 Dihydrothymine MedChemExpress Cat#HY-N6787 Dextran sulfate sodium (DSS) salt, colitis grade MP Biomedicals Cat#02160110-CF Dimethyl sulfoxide (DMSO) Sigma-Aldrich Cat#D4540 Glycine-b-muricholic acid MedChemExpress Cat#HY-114392 L-Histidine MedChemExpress Cat#HY-N0832 Lipofectamine 2000 Transfection Reagent Thermo Fisher Scientific Cat#11668019 Oleic acid MedChemExpress Cat#HY-N1446 Phosphate-buffered saline (PBS) tablets Thermo Fisher Scientific Cat#18912014 PI/RNase staining buffer BD Biosciences Cat#550825 SuperSignal West Femto Maximum Sensitivity Substrate Thermo Fisher Scientific Cat#34096 TRIzol Reagent Thermo Fisher Scientific Cat#15596026 Tween 20 Sigma-Aldrich Cat#P7949 Recombinant human ENO1 Cusabio Cat#CSB-YP007670HU Recombinant human FXR1 Cusabio Cat#CSB-EP009087HU(A4) Critical commercial assays Bio-Plex Pro Human Cytokine 27-plex Assay Bio-Rad Cat#M500KCAF0Y FITC Annexin V Apoptosis Detection Kit BD Biosciences Cat#556547; RRID: AB_2869082 OC-Sensor Fecal Test Eiken Chemical Cat#V-PZ25 Pierce BCA Protein Assay Kit Thermo Fisher Scientific Cat#23225 Pierce Quantitative Peptide Assays & Standards Thermo Fisher Scientific Cat#23275 PowerSoil Pro Kit Qiagen Cat#47017 Deposited data Metagenomic sequencing data of human fecal samples This paper CNGB: CNP0005876 Metabolomics of human plasma and fecal samples This paper CNGB: CNP0005882 RNA sequencing data of human CRC cells This paper NCBI BioProject: PRJNA1127337 Experimental models: cell lines Human: HCT116 cells ATCC CCL-247; RRID: CVCL_0291 Human: HT-29 cells ATCC HTB-38; RRID: CVCL_0320 Human: DLD-1 cells ATCC CCL-221; RRID: CVCL_0248 Human: 293T cells ATCC CRL-3216; RRID: CVCL_0063 Experimental models: organisms/strains Mouse: C57BL/6J The Chinese University of Hong Kong; strain purchased from The Jackson Laboratory Cat#000664; RRID: IMSR_JAX:000664 Mouse: C57BL/6J-ApcMin/+/J The Chinese University of Hong Kong; strain purchased from The Jackson Laboratory Cat#002020; RRID: IMSR_JAX:002020 Mouse: Nude The Chinese University of Hong Kong; strain purchased from The Jackson Laboratory Cat#002019; RRID: IMSR_JAX:002019 Oligonucleotides siRNA targeting sequence: Human ENO1-1 50-CAGUGGUGUCUAUCGAAGA-30 50-UCUUCGAUAGACACCACUG-30 Tsingke Biotechnology N/A (Continued on next page) e2 Cancer Cell 42, 1386–1400.e1–e8, August 12, 2024

Techniques: Binding Assay, SPR Assay, MANN-WHITNEY