Journal: MedComm

Article Title: PRMT5 promotes ovarian cancer growth through enhancing Warburg effect by methylating ENO1

doi: 10.1002/mco2.245

Figure Lengend Snippet: Targeting PRMT5 decreases ENO1 activity mediated by arginine methylation. (A) The protein expression of glycolytic enzymes was detected by western blotting in PRMT5 knockdown A2780 and OV2008 cells. (B) The interaction between glycolytic enzymes and PRMT5 was analyzed by Flag pull‐down assay. (C) The interaction between GST‐ENO1 and endogenous PRMT5 was analyzed by GST pull‐down assays in 293T cells. (D) GST‐ENO1 were pulled down from 293T cells that coexpressed Flag‐tagged PRMT5 or control vector, followed by ENO1 enzyme activity assay ( upper ) and western blotting ( lower ). (E) Recombinant PRMT5 and GST‐tagged ENO1 (rPRMT5 and rGST‐ENO1) purified from bacteria were incubated with each other in vitro. The interaction between ENO1 and PRMT5 was analyzed by Co‐IP. (F) A2780 extracts were immunoprecipitated with anti‐ENO1 or control rabbit IgG and western blotted by PRMT5 antibody. (G) Nucleocytoplasmic separation experiment was performed to detect PRMT5 and ENO1. (H) The endogenous enzymatic activity ( upper ) and SDMA level ( lower ) of ENO1 were analyzed in PRMT5 knockdown A2780 cells. (H, Heavy chain; E, Endogenous‐ENO1; L, Light chain.) (I) The endogenous enzymatic activity ( upper ) and SDMA level ( lower ) of ENO1 were analyzed in A2780 cells overexpressed PRMT5 WT or ΔM. (All error bars, mean values ± SD, p values were determined by unpaired two‐tailed Student's t test of n = 3 independent biological experiments. * p < 0.05; **p < 0.01; ***p < 0.001).

Article Snippet: Proteomics analysis (PhosphoSitePlus powered by Cell Signaling Technology) revealed that there was a group of arginine methylation residues on ENO1 protein ( https://www.phosphosite.org/proteinAction.action ?id = 2610&showAllSites = true and https://www.phosphosite.org/siteTableNewAction ?id = 2610&showAllSites = true ).

Techniques: Activity Assay, Methylation, Expressing, Western Blot, Pull Down Assay, Plasmid Preparation, Enzyme Activity Assay, Recombinant, Purification, Incubation, In Vitro, Co-Immunoprecipitation Assay, Immunoprecipitation, Two Tailed Test

Journal: MedComm

Article Title: PRMT5 promotes ovarian cancer growth through enhancing Warburg effect by methylating ENO1

doi: 10.1002/mco2.245

Figure Lengend Snippet: PRMT5 signals high glucose condition and to increase ENO1 activity by methylating its R9 site. (A) in vitro methylation assay was performed to detect the SDMA level of purified rGST‐ENO1, which was incubated with rPRMT5 wild type (WT) or inactivating mutant (ΔM). (B) The SDMA level of rGST‐ENO1 WT, R9K, R15K, R50K, R183K, R372K, and R412K were detected by western blotting. (C) The activity of purified rGST‐ENO1 WT and R9K was examined by ENO1 enzyme activity assay. (D) in vitro methylation assay was performed to detect the SDMA level of purified rGST‐ENO1, R9K, and R412K, which were incubated with or without rPRMT5 WT. (E) Structural location of R9 site on ENO1 was indicated (PDB ID 2PSN). (F) Crosslinking assay was performed to detect dimeric and monomeric ENO1 ( upper ; ENO1 antibody). ENO1 protein input assessed by western blotting is shown in the lower panel. (D, dimeric GST‐ENO1, 144 KD; M, monomeric GST‐ENO1, 72 KD). (G) Flag‐ENO1 WT, R9K, and R412K were expressed in 293T cells, followed by Flag pull‐down and western blotting to analyze the interaction between exogenous Flag‐ENO1 and endogenous ENO1. (H) Purified rGST‐ENO1 WT and R9K were incubated with or without rPRMT5 for in vitro methylation assay, followed by ENO1 enzymatic assays ( upper ), crosslinking assays ( middle ), and western blotting ( lower ). (I) The SDMA level of Flag‐ENO1 was detected by western blotting in A2780 cells, which treated with different concentrations of glucose for 12 h. (J) The activity and expression of ENO1 were detected by enzyme assay and western blotting in A2780 cells, which treated with different concentrations of glucose for 12 h. (K) The SDMA level of Flag‐ENO1 WT or ENO1 R9K was detected by western blotting in A2780 cells, which treated with or without 25 mM glucose for 12 h. (L) The activity ( left ) and SDMA level ( right ) of ENO1 were detected by enzyme assay and western blotting in PRMT5 knockdown or vector control A2780 cells, which treated with or without 25 mM glucose for 12 h. (I–L Before 0 or 25 mM sugar treatment, cells in each group were first subjected to glucose starvation for 6 h, and then normal cultured for 2 h. H, heavy chain; E, endogenous‐ENO1. All error bars, mean values ± SD, p values were determined by unpaired two‐tailed Student's t test of n = 3 independent biological experiments. *p < 0.05; **p < 0.01; ***p < 0.001).

Article Snippet: Proteomics analysis (PhosphoSitePlus powered by Cell Signaling Technology) revealed that there was a group of arginine methylation residues on ENO1 protein ( https://www.phosphosite.org/proteinAction.action ?id = 2610&showAllSites = true and https://www.phosphosite.org/siteTableNewAction ?id = 2610&showAllSites = true ).

Techniques: Activity Assay, In Vitro, Methylation, Purification, Incubation, Mutagenesis, Western Blot, Enzyme Activity Assay, Expressing, Enzymatic Assay, Plasmid Preparation, Cell Culture, Two Tailed Test

Journal: MedComm

Article Title: PRMT5 promotes ovarian cancer growth through enhancing Warburg effect by methylating ENO1

doi: 10.1002/mco2.245

Figure Lengend Snippet: PRMT5 promotes cancer cell growth and glycolysis flux through activating ENO1. (A) Cell proliferation was determined by cell number counting in A2780 and OVCAR3 cells with ENO1 knockdown. (B) Relative lactate level ( upper ) was determined by Llactic Acid assay kit in A2780 and OVCAR3 cells with ENO1 knockdown. (C) Cell proliferation was determined by cell number counting in A2780 and OVCAR3 cells with ENO1 knockdown, which exogenous express Flag‐ENO1 WT or R9K (empty vector worked as a control). (D) Relative lactate level ( upper ) was determined by Lactic Acid assay kit in A2780 and OVCAR3 cells with ENO1 knockdown, which exogenous express Flag‐ENO1 WT or R9K (empty vector worked as a control). (E) Cell proliferation was determined by cell number counting in A2780 and OVCAR3 cells with PRMT5 knockdown, which exogenously express Flag‐ENO1 WT or R9K (empty vector worked as a control). (F) Relative lactate level ( upper ) was determined by Lactic Acid assay kit in A2780 and OVCAR3 cells with PRMT5 knockdown, which exogenously express Flag‐ENO1 WT or R9K (empty vector worked as a control). (All error bars, mean values ± SD, p values were determined by unpaired two‐tailed Student's t test of n = 3 independent biological experiments. *p < 0.05; **p < 0.01; ***p < 0.001).

Article Snippet: Proteomics analysis (PhosphoSitePlus powered by Cell Signaling Technology) revealed that there was a group of arginine methylation residues on ENO1 protein ( https://www.phosphosite.org/proteinAction.action ?id = 2610&showAllSites = true and https://www.phosphosite.org/siteTableNewAction ?id = 2610&showAllSites = true ).

Techniques: Acid Assay, Plasmid Preparation, Two Tailed Test

Journal: MedComm

Article Title: PRMT5 promotes ovarian cancer growth through enhancing Warburg effect by methylating ENO1

doi: 10.1002/mco2.245

Figure Lengend Snippet: PRMT5 inhibitors reduce ENO1 enzymatic activity and glycolytic pathway. (A) The cell proliferation was determined by cell number counting in ovarian cancer cells treated with different concentrations of DW14761. (B) Colony formation assay was determined in ovarian cancer cells treated with different concentrations of DW14761. (C) The cell proliferation was determined by cell number counting in ovarian cancer cells treated with different concentrations of DW14800. (D) Colony formation assay was determined in ovarian cancer cells treated with different concentrations of DW14800. (E) The ECAR was measured in A2780 cells treated with different concentrations of DW14761. (2‐DG, 2‐Deoxy‐D‐glucose, the inhibitor of glycolytic pathway). (F) The ECAR was measured in OV2008 cells treated with different concentrations of DW14761. (G) The ECAR was measured in A2780 cells treated with different concentrations of DW14800. (H) The ECAR was measured in OV2008 cells treated with different concentrations of DW14800. (All error bars, mean values ± SD, p values were determined by unpaired two‐tailed Student's t test of n = 3 independent biological experiments. *p < 0.05; **p < 0.01; ***p < 0.001).

Article Snippet: Proteomics analysis (PhosphoSitePlus powered by Cell Signaling Technology) revealed that there was a group of arginine methylation residues on ENO1 protein ( https://www.phosphosite.org/proteinAction.action ?id = 2610&showAllSites = true and https://www.phosphosite.org/siteTableNewAction ?id = 2610&showAllSites = true ).

Techniques: Activity Assay, Colony Assay, Two Tailed Test

Journal: MedComm

Article Title: PRMT5 promotes ovarian cancer growth through enhancing Warburg effect by methylating ENO1

doi: 10.1002/mco2.245

Figure Lengend Snippet: DW14761 as a PRMT5 inhibitor inhibits SDMA level and enzymatic activity of ENO1 and ovarian cancer growth. (A) The protein expression of glycolytic enzymes was detected by western blotting in ovarian cancer cells treated with different concentrations of DW14761 and DW14800. (B) The endogenous enzymatic activity (upper) and SDMA level (lower) of ENO1 were analyzed in A2780 cells treated with different concentrations of DW14761. (H, Heavy chain; E, Endogenous‐ENO1). (C) The activity ( left ) and SDMA level ( right ) of ENO1 were detected by enzyme assay and western blotting in A2780 cells, which treated with or without 20 μM DW14761 for 36 h, then followed by treatment with or without 25 mM glucose for 12 h. (H, Heavy chain; E, Endogenous‐ENO1). (D) The SDMA level of ENO1 was detected by western blotting in OVCAR3 cells, which treated with or without 20 μM DW14761 for 36 h, and then followed by treatment with or without 25 mM glucose for 12 h. (E) Tumor growth in xenograft nude mice injected with A2780 cells was compared between the group of mice treated with DW14761 and the control group treated with vehicle control. (F) Tumor weights in xenograft nude mice injected with A2780 cells were compared between the group of mice treated with DW14761 and the control group treated with vehicle control. (G) All tumors in xenograft nude mouse were shown. (H) Mice body weights in each group were recorded. (I) IHC was performed to detect the Ki67 level in a representative tumor. (All error bars, mean values ± SD, p values were determined by unpaired two‐tailed Student's t test of n = 3 independent biological experiments. * p < 0.05; **p < 0.01; ***p < 0.001).

Article Snippet: Proteomics analysis (PhosphoSitePlus powered by Cell Signaling Technology) revealed that there was a group of arginine methylation residues on ENO1 protein ( https://www.phosphosite.org/proteinAction.action ?id = 2610&showAllSites = true and https://www.phosphosite.org/siteTableNewAction ?id = 2610&showAllSites = true ).

Techniques: Activity Assay, Expressing, Western Blot, Enzymatic Assay, Injection, Two Tailed Test

Journal: Molecules

Article Title: Oridonin Induces Apoptosis in Esophageal Squamous Cell Carcinoma by Inhibiting Cytoskeletal Protein LASP1 and PDLIM1

doi: 10.3390/molecules28020805

Figure Lengend Snippet: Top Candidate Proteins Involved in Oridonin-treated ESCC.

Article Snippet: Membranes were blocked with 5% Beef Serum Albumin (BSA) and then incubated with primary antibodies: mouse Hsp70 antibody (mAb; Abcam, Boston, MA, USA), rabbit ENO1, LASP1, GAPDH antibodies (#3810 (pAb), #8636 (pAb), #2218 (mAb); cell signaling, Danvers, MA, USA), rabbit PDLIM1 antibody (ABclonal, Cambridge, MA, USA), and mouse β -Actin antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA).

Techniques: Variant Assay

Journal: Molecules

Article Title: Oridonin Induces Apoptosis in Esophageal Squamous Cell Carcinoma by Inhibiting Cytoskeletal Protein LASP1 and PDLIM1

doi: 10.3390/molecules28020805

Figure Lengend Snippet: Gene Set Enrichment Analysis and western blotting assessed that oridonin inhibits LASP1 and PDLIM1 on ESCC. ( A ) Molecular function classification of the protein candidates involved in oridonin treatment. The top three are cadherin binding, cadherin binding involved in cell–cell adhesion, and heat shock protein binding. ( B ) The top three cellular component classifications are focal adhesion, cell-substrate junction, and cell cortex. ( C ) The top three biological processes are negative regulation of apoptotic signaling pathway, removal of superoxide radicals, and cellular response to oxygen radicals. ( D ) Protein expression of LASP1 and PDLIM1 under oridonin treatment decreased compared to untreated cell lysates. Hsp70 expression increased in oridonin-treated cell lysates compared to untreated while ENO1 decreased a little in treated TE-8.

Article Snippet: Membranes were blocked with 5% Beef Serum Albumin (BSA) and then incubated with primary antibodies: mouse Hsp70 antibody (mAb; Abcam, Boston, MA, USA), rabbit ENO1, LASP1, GAPDH antibodies (#3810 (pAb), #8636 (pAb), #2218 (mAb); cell signaling, Danvers, MA, USA), rabbit PDLIM1 antibody (ABclonal, Cambridge, MA, USA), and mouse β -Actin antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA).

Techniques: Western Blot, Binding Assay, Protein Binding, Expressing

Journal: Molecules

Article Title: Oridonin Induces Apoptosis in Esophageal Squamous Cell Carcinoma by Inhibiting Cytoskeletal Protein LASP1 and PDLIM1

doi: 10.3390/molecules28020805

Figure Lengend Snippet: Interested Genes for Protein Assessment Involved in Oridonin Treatment.

Article Snippet: Membranes were blocked with 5% Beef Serum Albumin (BSA) and then incubated with primary antibodies: mouse Hsp70 antibody (mAb; Abcam, Boston, MA, USA), rabbit ENO1, LASP1, GAPDH antibodies (#3810 (pAb), #8636 (pAb), #2218 (mAb); cell signaling, Danvers, MA, USA), rabbit PDLIM1 antibody (ABclonal, Cambridge, MA, USA), and mouse β -Actin antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA).

Techniques: Binding Assay, Protein Binding

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Mediation of PKM2-dependent glycolytic and non-glycolytic pathways by ENO2 in head and neck cancer development

doi: 10.1186/s13046-022-02574-0

Figure Lengend Snippet: AP-III-a4 inhibits cell proliferation and glucose metabolism in HNSCC cells through suppressing ENO2-mediated downstream signaling. a Effect of AP-III-a4 treatment on ENO1 and ENO2 protein levels determined by western blotting. UM-1 and Cal27 cells were treated with AP-III-a4 at a dose range (0, 1, 2.5, 5µM) for 48 h. b - e Effect of AP-III-a4 treatment on ENO2-mediated downstream targets ( b ), cell proliferation ( c ), and intracellular levels of ATP ( d ) and glucose uptake ( e ). UM-1 and Cal27 cells were treated with or without 5µM AP-III-a4 for 48 h. * p <0.05; ** p <0.01

Article Snippet: ERK1/2, p-ERK1/2, AKT, p-AKT, P70S6K, p-P70S6K, cyclin D1 (CCND1), cyclin B, cyclin E, CDK2, CDK4, CDK6 and ENO1 antibodies were purchased from Cell Signaling Technology.

Techniques: Western Blot