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