recombinant human eno1  (Cusabio)

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

Journal: Frontiers in Oncology

Article Title: ENO1 contributes to 5-fluorouracil resistance in colorectal cancer cells via EMT pathway

doi: 10.3389/fonc.2022.1013035

Figure Lengend Snippet: Expression of ENO1 in CRC tissues. (A) Representative immunohistochemical (IHC) images showing in situ ENO1 expression in colorectal cancer (CRC) and normal tissues (scale bar = 100 μm). (B) IHC scores of ENO1 in CRC vs normal tissues. (C) IHC scores of ENO1 in T I-II vs T III-IV tissues. (D–F) Overall survival of CRC patients with high and low expressions (D) of ENO1 with TNM staging I-II (E) and III-IV (F) . Statistical analyses were performed via two-tailed Student t test. ****, P ≤ 0.001.

Article Snippet: The sections were incubated with 1:100 diluted polyclonal anti-human ENO1 (BOSTER, Wuhan, China) at 4°C overnight.

Techniques: Expressing, Immunohistochemical staining, In Situ, Two Tailed Test

Journal: Frontiers in Oncology

Article Title: ENO1 contributes to 5-fluorouracil resistance in colorectal cancer cells via EMT pathway

doi: 10.3389/fonc.2022.1013035

Figure Lengend Snippet: Relationship between ENO1 and clinic-pathological factors in CRC patients.

Article Snippet: The sections were incubated with 1:100 diluted polyclonal anti-human ENO1 (BOSTER, Wuhan, China) at 4°C overnight.

Techniques: Expressing

Journal: Frontiers in Oncology

Article Title: ENO1 contributes to 5-fluorouracil resistance in colorectal cancer cells via EMT pathway

doi: 10.3389/fonc.2022.1013035

Figure Lengend Snippet: Results of univariate and multivariate analyses of postoperative patients’ survival by Cox’s proportional hazard model.

Article Snippet: The sections were incubated with 1:100 diluted polyclonal anti-human ENO1 (BOSTER, Wuhan, China) at 4°C overnight.

Techniques:

Journal: Frontiers in Oncology

Article Title: ENO1 contributes to 5-fluorouracil resistance in colorectal cancer cells via EMT pathway

doi: 10.3389/fonc.2022.1013035

Figure Lengend Snippet: Subgroup analysis for the influencing factor of survival among colorectal cancer patients according to ENO1 expression. Statistical analyses were performed via two-tailed Student t test. *, P ≤ 0.05.

Article Snippet: The sections were incubated with 1:100 diluted polyclonal anti-human ENO1 (BOSTER, Wuhan, China) at 4°C overnight.

Techniques: Expressing, Two Tailed Test

Journal: Frontiers in Oncology

Article Title: ENO1 contributes to 5-fluorouracil resistance in colorectal cancer cells via EMT pathway

doi: 10.3389/fonc.2022.1013035

Figure Lengend Snippet: ENO1 expression was increased in the constructed 5-fluorouracil (FU)-resistant cells. (A) Comparison of ENO1 expression between various CRC cell lines in Cancer Cell Line Encyclopedia (CCLE). (B, C) Immunoblotting results of ENO1 (B) in cell lines HCT116, SW620, HT29, SW480, and LOVO and quantitative counting (C) . (D) Cell viability comparison between HCT116 cells and 5-FU-resistant cells. (E, F) Increase in ENO1 expression (E) in 5-FU-resistant cells and the statistical graph (F) . Statistical analyses were performed via two-tailed Student t test. *,P ≤ 0.05; **,P ≤ 0.01.

Article Snippet: The sections were incubated with 1:100 diluted polyclonal anti-human ENO1 (BOSTER, Wuhan, China) at 4°C overnight.

Techniques: Expressing, Construct, Comparison, Western Blot, Two Tailed Test

Journal: Frontiers in Oncology

Article Title: ENO1 contributes to 5-fluorouracil resistance in colorectal cancer cells via EMT pathway

doi: 10.3389/fonc.2022.1013035

Figure Lengend Snippet: Knockdown of ENO1 inhibited the proliferation of 5-fluorouracil (FU)-resistant colorectal cancer (CRC) cells. (A, B) Immunoblots (A) showing ENO1 protein levels in NC and ENO1-KD group and the quantified pattern (B) , reflecting the success of transfection. (C, D) Cell viability of HCT/5-FU (C) and SW620/5-FU (D) cells in the ENO1-KD group and NC group. (E, F) Proliferation rates (E) and colony forming capacity (F) of 5-FU-resistant CRC cells in the ENO1-KD and NC groups (scale bar = 6mm). Statistical analyses were performed via two-tailed Student t test. *,P ≤ 0.05; **,P ≤ 0.01; ***,P ≤ 0.001.

Article Snippet: The sections were incubated with 1:100 diluted polyclonal anti-human ENO1 (BOSTER, Wuhan, China) at 4°C overnight.

Techniques: Knockdown, Western Blot, Transfection, Two Tailed Test

Journal: Frontiers in Oncology

Article Title: ENO1 contributes to 5-fluorouracil resistance in colorectal cancer cells via EMT pathway

doi: 10.3389/fonc.2022.1013035

Figure Lengend Snippet: ENO1 knockdown inhibited the migration of resistant cells and was associated with the EMT process. (A–C) Display of representative photographs (A) in the ENO1-KD and NC groups and the number of migration cells in HCT116/5-FU cells (scale bar = 100 μm) (B) and SW620/5-FU cells (C) . (D, F) Western blotting of E-Cadherin, N-Cadherin, and Vimentin expression levels following knockdown of ENO1 in HCT116/5-FU cells (D) and SW620/5-FU cells (F) . (E, G) The bands were quantified and are presented as mean ± SEM of three independent experiments in HCT116/5-FU cells (E) and SW620/5-FU cells (G) . Statistical analyses were performed via two-tailed Student t test. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.

Article Snippet: The sections were incubated with 1:100 diluted polyclonal anti-human ENO1 (BOSTER, Wuhan, China) at 4°C overnight.

Techniques: Knockdown, Migration, Western Blot, Expressing, Two Tailed Test

Journal: Frontiers in Oncology

Article Title: ENO1 contributes to 5-fluorouracil resistance in colorectal cancer cells via EMT pathway

doi: 10.3389/fonc.2022.1013035

Figure Lengend Snippet: The regulation of the EMT process by ENO1 is associated with energy metabolism. (A) The morphological changes during the EMT process were suppressed in the ENO1-KD group compared with those in the NC group (observed by light microscope at 200× magnification). (B, C) Western blotting of E-Cadherin, N-Cadherin, and Vimentin expression in NC and ENO1-KD groups treated with or without 20 mM 2-DG for 4 h in HCT116/5-FU cells (B) and SW620/5-FU cells (C) . (D-E) The bands were quantified and are presented as mean ± SEM of three independent experiments in HCT116/5-FU cells (D) and SW620/5-FU cells (E) . Statistical analyses were performed via two-tailed Student t test. **, P ≤ 0.01; ***, P ≤ 0.001. ns, no significance.

Article Snippet: The sections were incubated with 1:100 diluted polyclonal anti-human ENO1 (BOSTER, Wuhan, China) at 4°C overnight.

Techniques: Light Microscopy, Western Blot, Expressing, Two Tailed Test

Journal: Frontiers in Oncology

Article Title: ENO1 contributes to 5-fluorouracil resistance in colorectal cancer cells via EMT pathway

doi: 10.3389/fonc.2022.1013035

Figure Lengend Snippet: ENO1 knockdown suppresses tumor growth in colorectal cancer (CRC) in vivo . (A, B) Total body weight and tumor volume in ENO1-KD and NC groups. (C, D) Total body weight and tumor volume in ENO1-KD+5-FU and 5-FU groups. (E) Representative pictures of subcutaneous tumors harvested from the four groups. (F) The weights of tumor masses with or without treatment. (G) Stratification of mice into cluster 1 (grey) and cluster 2 (blue) according to tumor weight and ENO1 levels. (H) Percentage of NC and ENO1-KD mice in each cluster. Statistical analyses were performed via two-tailed Student t test. *, P ≤ 0.05; **, P ≤ 0.01.

Article Snippet: The sections were incubated with 1:100 diluted polyclonal anti-human ENO1 (BOSTER, Wuhan, China) at 4°C overnight.

Techniques: Knockdown, In Vivo, Two Tailed Test