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Imbalance of Th17/Treg immune cells, NF‐κB signal transduction, and pyruvate metabolic process were associated with the occurrence and development of IBD. (A) UMAP visualization of cells from healthy and IBD; (B) Cell type distribution in UMAP after manual annotation of each cluster based on known marker genes; (C) Top4 characteristic genes with the highest expression levels within each cell population; (D) Stacked bar chart showing the relative abundance of each cell type in healthy and IBD; (E) Stacked bar chart of the proportion of each subpopulation after further subdivision of T cell population; (F, G) UMAP feature maps showing the expression distribution of RELA (encoding p65) and <t>NLRP3;</t> (H, I) Expression levels of RELA and NLRP3 across different cell types; (J, K) GSEA results focused on the pyruvate metabolism and NF‐κB pathways.
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Imbalance of Th17/Treg immune cells, NF‐κB signal transduction, and pyruvate metabolic process were associated with the occurrence and development of IBD. (A) UMAP visualization of cells from healthy and IBD; (B) Cell type distribution in UMAP after manual annotation of each cluster based on known marker genes; (C) Top4 characteristic genes with the highest expression levels within each cell population; (D) Stacked bar chart showing the relative abundance of each cell type in healthy and IBD; (E) Stacked bar chart of the proportion of each subpopulation after further subdivision of T cell population; (F, G) UMAP feature maps showing the expression distribution of RELA (encoding p65) and <t>NLRP3;</t> (H, I) Expression levels of RELA and NLRP3 across different cell types; (J, K) GSEA results focused on the pyruvate metabolism and NF‐κB pathways.
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Imbalance of Th17/Treg immune cells, NF‐κB signal transduction, and pyruvate metabolic process were associated with the occurrence and development of IBD. (A) UMAP visualization of cells from healthy and IBD; (B) Cell type distribution in UMAP after manual annotation of each cluster based on known marker genes; (C) Top4 characteristic genes with the highest expression levels within each cell population; (D) Stacked bar chart showing the relative abundance of each cell type in healthy and IBD; (E) Stacked bar chart of the proportion of each subpopulation after further subdivision of T cell population; (F, G) UMAP feature maps showing the expression distribution of RELA (encoding p65) and <t>NLRP3;</t> (H, I) Expression levels of RELA and NLRP3 across different cell types; (J, K) GSEA results focused on the pyruvate metabolism and NF‐κB pathways.
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Imbalance of Th17/Treg immune cells, NF‐κB signal transduction, and pyruvate metabolic process were associated with the occurrence and development of IBD. (A) UMAP visualization of cells from healthy and IBD; (B) Cell type distribution in UMAP after manual annotation of each cluster based on known marker genes; (C) Top4 characteristic genes with the highest expression levels within each cell population; (D) Stacked bar chart showing the relative abundance of each cell type in healthy and IBD; (E) Stacked bar chart of the proportion of each subpopulation after further subdivision of T cell population; (F, G) UMAP feature maps showing the expression distribution of RELA (encoding p65) and NLRP3; (H, I) Expression levels of RELA and NLRP3 across different cell types; (J, K) GSEA results focused on the pyruvate metabolism and NF‐κB pathways.

Journal: Advanced Science

Article Title: Uhrf1‐Mediated PKM2 Degradation via Ubiquitination Alleviates Inflammation and Pyroptosis in Inflammatory Bowel Disease

doi: 10.1002/advs.77403

Figure Lengend Snippet: Imbalance of Th17/Treg immune cells, NF‐κB signal transduction, and pyruvate metabolic process were associated with the occurrence and development of IBD. (A) UMAP visualization of cells from healthy and IBD; (B) Cell type distribution in UMAP after manual annotation of each cluster based on known marker genes; (C) Top4 characteristic genes with the highest expression levels within each cell population; (D) Stacked bar chart showing the relative abundance of each cell type in healthy and IBD; (E) Stacked bar chart of the proportion of each subpopulation after further subdivision of T cell population; (F, G) UMAP feature maps showing the expression distribution of RELA (encoding p65) and NLRP3; (H, I) Expression levels of RELA and NLRP3 across different cell types; (J, K) GSEA results focused on the pyruvate metabolism and NF‐κB pathways.

Article Snippet: The primary antibodies occludin (27260‐1‐AP), RORγt (29910‐1‐AP), Foxp3 (22228‐1‐AP), GSDMD (66387‐1‐Ig), LDHA (19987‐1‐AP), GLUT1 (21829‐1‐AP), Mfn1 (13798‐1‐AP), Mfn2 (12186‐1‐AP), Opa1 (27733‐1‐AP), Fis1 (10956‐1‐AP), TOM20 (A19403), COX‐2 (66351‐1‐Ig), iNOS (22226‐1‐AP), p‐p65 (AP0124), p65 (10745‐1‐AP), caspase 1 (22915‐1‐AP), IL‐1β (26048‐1‐AP), ASC ( AB309497 ), NLRP3 (68102‐1‐Ig), PKM2 (60268‐1‐Ig), Drp1 (12957‐1‐AP), H3 (17168‐1‐AP), HA (51054‐1‐AP), GST (80006‐1‐AP), HIS (66005‐1‐AP), Ub (10201‐1‐AP), Uhrf1 (21402‐1‐AP), and GAPDH (60004‐1‐Ig) were purchased from Abcam (Shanghai, China), Abclonal (Wuhan, China) and Proteintech (Wuhan, China), respectively.

Techniques: Transduction, Marker, Expressing

PKM2 genetic knockdown abolished the anti‐IBD effect of EPT in vivo. (A) Schematic diagram for PKM2 knockdown experiments; (B) Body weight, diarrhea and DAI score ( n = 8); (C) H&E staining plot; (D) Colonoscopy plots; (E) Gross colon morphology and length ( n = 6); (F and I) PKM2 KO abolished effects of EPT toward mRNA levels of IL‐1β , CCL5 , IL‐6 , IL‐1α , CXCL10 , NLRP3, Foxp3 , and RORc ( n = 3); (G, H) Flow cytometry results of Th17 (CD4 + IL‐17A + ) and Treg (CD4 + CD25 + Foxp3 + ) cells ( n = 3); (J, K) RORγt/Foxp3 and iNOS/NLRP3 staining plots; (L) PKM2 knockdown abolished effects of EPT toward expressions of RORγt, Foxp3, iNOS, p‐p65, p65, IL‐1β, pro IL‐1β, GSDMD‐N, GSDMD, and NLRP3.

Journal: Advanced Science

Article Title: Uhrf1‐Mediated PKM2 Degradation via Ubiquitination Alleviates Inflammation and Pyroptosis in Inflammatory Bowel Disease

doi: 10.1002/advs.77403

Figure Lengend Snippet: PKM2 genetic knockdown abolished the anti‐IBD effect of EPT in vivo. (A) Schematic diagram for PKM2 knockdown experiments; (B) Body weight, diarrhea and DAI score ( n = 8); (C) H&E staining plot; (D) Colonoscopy plots; (E) Gross colon morphology and length ( n = 6); (F and I) PKM2 KO abolished effects of EPT toward mRNA levels of IL‐1β , CCL5 , IL‐6 , IL‐1α , CXCL10 , NLRP3, Foxp3 , and RORc ( n = 3); (G, H) Flow cytometry results of Th17 (CD4 + IL‐17A + ) and Treg (CD4 + CD25 + Foxp3 + ) cells ( n = 3); (J, K) RORγt/Foxp3 and iNOS/NLRP3 staining plots; (L) PKM2 knockdown abolished effects of EPT toward expressions of RORγt, Foxp3, iNOS, p‐p65, p65, IL‐1β, pro IL‐1β, GSDMD‐N, GSDMD, and NLRP3.

Article Snippet: The primary antibodies occludin (27260‐1‐AP), RORγt (29910‐1‐AP), Foxp3 (22228‐1‐AP), GSDMD (66387‐1‐Ig), LDHA (19987‐1‐AP), GLUT1 (21829‐1‐AP), Mfn1 (13798‐1‐AP), Mfn2 (12186‐1‐AP), Opa1 (27733‐1‐AP), Fis1 (10956‐1‐AP), TOM20 (A19403), COX‐2 (66351‐1‐Ig), iNOS (22226‐1‐AP), p‐p65 (AP0124), p65 (10745‐1‐AP), caspase 1 (22915‐1‐AP), IL‐1β (26048‐1‐AP), ASC ( AB309497 ), NLRP3 (68102‐1‐Ig), PKM2 (60268‐1‐Ig), Drp1 (12957‐1‐AP), H3 (17168‐1‐AP), HA (51054‐1‐AP), GST (80006‐1‐AP), HIS (66005‐1‐AP), Ub (10201‐1‐AP), Uhrf1 (21402‐1‐AP), and GAPDH (60004‐1‐Ig) were purchased from Abcam (Shanghai, China), Abclonal (Wuhan, China) and Proteintech (Wuhan, China), respectively.

Techniques: Knockdown, In Vivo, Staining, Flow Cytometry

EPT regulated the balance of Th17/Treg immune cells and inflammation to alleviate the course of DSS‐mediated IBD in a mouse model. (A) Structure of EPT; (B) Timeline for the DSS‐mediated IBD experiment; (C) Body weight, diarrhea score, and DAI score ( n = 8); (D) Gross colon morphology and length ( n = 6); (E) Colonoscopy plots; (F) H&E and occludin staining plots; (G) Effects of EPT toward mRNA levels of IL‐6 and IL‐1β ( n = 3); (H) RORγt/Foxp3 staining plots; (I) Effects of EPT toward expressions for RORγt and Foxp3 in spleen ( n = 3); (J) Flow cytometry results for Th17 (CD4 + IL‐17A + ) and Treg (CD4 + CD25 + Foxp3 + ) cells ( n = 3) after defining the gate of CD4 + T cells by FITC‐CD4 anti‐body; (K) EPT regulated expressions of RORγt and Foxp3, and suppressed NF‐κB and NLRP3 pathways involved in iNOS, p‐p65, p65, IL‐1β, pro IL‐1β, cleaved caspase 1, caspase 1, GSDMD‐N, GSDMD, ASC, and NLRP3.

Journal: Advanced Science

Article Title: Uhrf1‐Mediated PKM2 Degradation via Ubiquitination Alleviates Inflammation and Pyroptosis in Inflammatory Bowel Disease

doi: 10.1002/advs.77403

Figure Lengend Snippet: EPT regulated the balance of Th17/Treg immune cells and inflammation to alleviate the course of DSS‐mediated IBD in a mouse model. (A) Structure of EPT; (B) Timeline for the DSS‐mediated IBD experiment; (C) Body weight, diarrhea score, and DAI score ( n = 8); (D) Gross colon morphology and length ( n = 6); (E) Colonoscopy plots; (F) H&E and occludin staining plots; (G) Effects of EPT toward mRNA levels of IL‐6 and IL‐1β ( n = 3); (H) RORγt/Foxp3 staining plots; (I) Effects of EPT toward expressions for RORγt and Foxp3 in spleen ( n = 3); (J) Flow cytometry results for Th17 (CD4 + IL‐17A + ) and Treg (CD4 + CD25 + Foxp3 + ) cells ( n = 3) after defining the gate of CD4 + T cells by FITC‐CD4 anti‐body; (K) EPT regulated expressions of RORγt and Foxp3, and suppressed NF‐κB and NLRP3 pathways involved in iNOS, p‐p65, p65, IL‐1β, pro IL‐1β, cleaved caspase 1, caspase 1, GSDMD‐N, GSDMD, ASC, and NLRP3.

Article Snippet: The primary antibodies occludin (27260‐1‐AP), RORγt (29910‐1‐AP), Foxp3 (22228‐1‐AP), GSDMD (66387‐1‐Ig), LDHA (19987‐1‐AP), GLUT1 (21829‐1‐AP), Mfn1 (13798‐1‐AP), Mfn2 (12186‐1‐AP), Opa1 (27733‐1‐AP), Fis1 (10956‐1‐AP), TOM20 (A19403), COX‐2 (66351‐1‐Ig), iNOS (22226‐1‐AP), p‐p65 (AP0124), p65 (10745‐1‐AP), caspase 1 (22915‐1‐AP), IL‐1β (26048‐1‐AP), ASC ( AB309497 ), NLRP3 (68102‐1‐Ig), PKM2 (60268‐1‐Ig), Drp1 (12957‐1‐AP), H3 (17168‐1‐AP), HA (51054‐1‐AP), GST (80006‐1‐AP), HIS (66005‐1‐AP), Ub (10201‐1‐AP), Uhrf1 (21402‐1‐AP), and GAPDH (60004‐1‐Ig) were purchased from Abcam (Shanghai, China), Abclonal (Wuhan, China) and Proteintech (Wuhan, China), respectively.

Techniques: Staining, Flow Cytometry

EPT suppressed NF‐κB‐mediated inflammation and NLRP3‐mediated pyroptosis in vitro. (A) Effects of EPT toward inflammatory factors NO, TNF‐α, and IL‐6 ( n = 3); (B) Effect of EPT toward lactic acid ( n = 3); (C) Flow cytometry results of calcium ion (Ca + , Fluo‐4, n = 3); (D) Heatmap of mRNA levels for iNOS , COX‐2 , IL‐1α , IL‐6 , CCL5 , Mfn1 , Mfn2 , and Opa1 ( n = 3); (E) Effects of EPT toward mRNA levels for LDHA, HK2, GLUT1 , and IL‐1β ( n = 3); (F) Flow cytometry results of IL‐1β and NLRP3 ( n = 3); (G) Effects of EPT toward expressions of proteins involved in mitochondrion, lactic acid metabolism, NF‐κB, and NLRP3 pathways; (H, I) p65/COX‐2 and NLRP3/GSDMD‐N immunofluorescence staining plots.

Journal: Advanced Science

Article Title: Uhrf1‐Mediated PKM2 Degradation via Ubiquitination Alleviates Inflammation and Pyroptosis in Inflammatory Bowel Disease

doi: 10.1002/advs.77403

Figure Lengend Snippet: EPT suppressed NF‐κB‐mediated inflammation and NLRP3‐mediated pyroptosis in vitro. (A) Effects of EPT toward inflammatory factors NO, TNF‐α, and IL‐6 ( n = 3); (B) Effect of EPT toward lactic acid ( n = 3); (C) Flow cytometry results of calcium ion (Ca + , Fluo‐4, n = 3); (D) Heatmap of mRNA levels for iNOS , COX‐2 , IL‐1α , IL‐6 , CCL5 , Mfn1 , Mfn2 , and Opa1 ( n = 3); (E) Effects of EPT toward mRNA levels for LDHA, HK2, GLUT1 , and IL‐1β ( n = 3); (F) Flow cytometry results of IL‐1β and NLRP3 ( n = 3); (G) Effects of EPT toward expressions of proteins involved in mitochondrion, lactic acid metabolism, NF‐κB, and NLRP3 pathways; (H, I) p65/COX‐2 and NLRP3/GSDMD‐N immunofluorescence staining plots.

Article Snippet: The primary antibodies occludin (27260‐1‐AP), RORγt (29910‐1‐AP), Foxp3 (22228‐1‐AP), GSDMD (66387‐1‐Ig), LDHA (19987‐1‐AP), GLUT1 (21829‐1‐AP), Mfn1 (13798‐1‐AP), Mfn2 (12186‐1‐AP), Opa1 (27733‐1‐AP), Fis1 (10956‐1‐AP), TOM20 (A19403), COX‐2 (66351‐1‐Ig), iNOS (22226‐1‐AP), p‐p65 (AP0124), p65 (10745‐1‐AP), caspase 1 (22915‐1‐AP), IL‐1β (26048‐1‐AP), ASC ( AB309497 ), NLRP3 (68102‐1‐Ig), PKM2 (60268‐1‐Ig), Drp1 (12957‐1‐AP), H3 (17168‐1‐AP), HA (51054‐1‐AP), GST (80006‐1‐AP), HIS (66005‐1‐AP), Ub (10201‐1‐AP), Uhrf1 (21402‐1‐AP), and GAPDH (60004‐1‐Ig) were purchased from Abcam (Shanghai, China), Abclonal (Wuhan, China) and Proteintech (Wuhan, China), respectively.

Techniques: In Vitro, Flow Cytometry, Immunofluorescence, Staining

PKM2 knockdown or overexpression revoked protective effects of EPT in vitro. (A) PKM2 knockdown verification ( n = 3); (B) PKM2 knockdown abolished effects of EPT toward mRNA levels of IL‐1β , IL‐1α , IL‐6 , Opa1 , Drp1 , LDHA , HK2 , and GLUT1 ( n = 3); (C) PKM2 knockdown abolished effects of EPT toward expression of iNOS, p‐p65, p65, and NLRP3; (D) p65, NLRP3, and GSDMD‐N staining plots; (E) PKM2 overexpression verification ( n = 3); (F) PKM2 overexpression weakened effects of EPT toward mRNA levels of IL‐6 , IL‐1α , CXCL10 , and IL‐1β ( n = 3); (G) PKM2 overexpression weakened effects of EPT toward expression of Drp1, Mfn1, p‐p65, p65, and NLRP3; (H) COX‐2 staining plots.

Journal: Advanced Science

Article Title: Uhrf1‐Mediated PKM2 Degradation via Ubiquitination Alleviates Inflammation and Pyroptosis in Inflammatory Bowel Disease

doi: 10.1002/advs.77403

Figure Lengend Snippet: PKM2 knockdown or overexpression revoked protective effects of EPT in vitro. (A) PKM2 knockdown verification ( n = 3); (B) PKM2 knockdown abolished effects of EPT toward mRNA levels of IL‐1β , IL‐1α , IL‐6 , Opa1 , Drp1 , LDHA , HK2 , and GLUT1 ( n = 3); (C) PKM2 knockdown abolished effects of EPT toward expression of iNOS, p‐p65, p65, and NLRP3; (D) p65, NLRP3, and GSDMD‐N staining plots; (E) PKM2 overexpression verification ( n = 3); (F) PKM2 overexpression weakened effects of EPT toward mRNA levels of IL‐6 , IL‐1α , CXCL10 , and IL‐1β ( n = 3); (G) PKM2 overexpression weakened effects of EPT toward expression of Drp1, Mfn1, p‐p65, p65, and NLRP3; (H) COX‐2 staining plots.

Article Snippet: The primary antibodies occludin (27260‐1‐AP), RORγt (29910‐1‐AP), Foxp3 (22228‐1‐AP), GSDMD (66387‐1‐Ig), LDHA (19987‐1‐AP), GLUT1 (21829‐1‐AP), Mfn1 (13798‐1‐AP), Mfn2 (12186‐1‐AP), Opa1 (27733‐1‐AP), Fis1 (10956‐1‐AP), TOM20 (A19403), COX‐2 (66351‐1‐Ig), iNOS (22226‐1‐AP), p‐p65 (AP0124), p65 (10745‐1‐AP), caspase 1 (22915‐1‐AP), IL‐1β (26048‐1‐AP), ASC ( AB309497 ), NLRP3 (68102‐1‐Ig), PKM2 (60268‐1‐Ig), Drp1 (12957‐1‐AP), H3 (17168‐1‐AP), HA (51054‐1‐AP), GST (80006‐1‐AP), HIS (66005‐1‐AP), Ub (10201‐1‐AP), Uhrf1 (21402‐1‐AP), and GAPDH (60004‐1‐Ig) were purchased from Abcam (Shanghai, China), Abclonal (Wuhan, China) and Proteintech (Wuhan, China), respectively.

Techniques: Knockdown, Over Expression, In Vitro, Expressing, Staining

EPT specifically modified amino acid residue C165 of PKM2. (A) IAA disrupted the binding affinity between EPT and PKM2; (B) Schematic diagram of the covalently binding identification of EPT with PKM2; (C) LC‐MS/MS plots; (D) Cys165Ala (C165A) mutation weakened the binding of EPT with PKM2 rather than Cys474Ala (C474A) mutation; (E) C165A mutation abolished anti‐inflammatory effects of EPT rather than C474A mutation in vitro ( n = 3); (F) MST plot of EPT with PKM2 C165A; (G) 3D interaction of EPT with PKM2; (H) PKM2 C165A recue enhanced mRNA levels of IL‐1β , IL‐6 , and CXCL10 in LPS‐mediated PKM2 knockdown cells ( n = 3); (I) PKM2 C165A recue activated NF‐κB and NLRP3 pathways in LPS or LPS plus NIG‐mediated PKM2 knockdown cells.

Journal: Advanced Science

Article Title: Uhrf1‐Mediated PKM2 Degradation via Ubiquitination Alleviates Inflammation and Pyroptosis in Inflammatory Bowel Disease

doi: 10.1002/advs.77403

Figure Lengend Snippet: EPT specifically modified amino acid residue C165 of PKM2. (A) IAA disrupted the binding affinity between EPT and PKM2; (B) Schematic diagram of the covalently binding identification of EPT with PKM2; (C) LC‐MS/MS plots; (D) Cys165Ala (C165A) mutation weakened the binding of EPT with PKM2 rather than Cys474Ala (C474A) mutation; (E) C165A mutation abolished anti‐inflammatory effects of EPT rather than C474A mutation in vitro ( n = 3); (F) MST plot of EPT with PKM2 C165A; (G) 3D interaction of EPT with PKM2; (H) PKM2 C165A recue enhanced mRNA levels of IL‐1β , IL‐6 , and CXCL10 in LPS‐mediated PKM2 knockdown cells ( n = 3); (I) PKM2 C165A recue activated NF‐κB and NLRP3 pathways in LPS or LPS plus NIG‐mediated PKM2 knockdown cells.

Article Snippet: The primary antibodies occludin (27260‐1‐AP), RORγt (29910‐1‐AP), Foxp3 (22228‐1‐AP), GSDMD (66387‐1‐Ig), LDHA (19987‐1‐AP), GLUT1 (21829‐1‐AP), Mfn1 (13798‐1‐AP), Mfn2 (12186‐1‐AP), Opa1 (27733‐1‐AP), Fis1 (10956‐1‐AP), TOM20 (A19403), COX‐2 (66351‐1‐Ig), iNOS (22226‐1‐AP), p‐p65 (AP0124), p65 (10745‐1‐AP), caspase 1 (22915‐1‐AP), IL‐1β (26048‐1‐AP), ASC ( AB309497 ), NLRP3 (68102‐1‐Ig), PKM2 (60268‐1‐Ig), Drp1 (12957‐1‐AP), H3 (17168‐1‐AP), HA (51054‐1‐AP), GST (80006‐1‐AP), HIS (66005‐1‐AP), Ub (10201‐1‐AP), Uhrf1 (21402‐1‐AP), and GAPDH (60004‐1‐Ig) were purchased from Abcam (Shanghai, China), Abclonal (Wuhan, China) and Proteintech (Wuhan, China), respectively.

Techniques: Modification, Residue, Binding Assay, Liquid Chromatography with Mass Spectroscopy, Mutagenesis, In Vitro, Knockdown

Anti‐inflammatory effects of EPT depended on Uhrf1‐mediated PKM2 ubiquitination. (A) Uhrf1 knockdown verification ( n = 3); (B) Uhrf1 knockdown weakened effects of EPT toward mRNA levels of IL‐1β , iNOS , and COX‐2 in PKM2 overexpression cells ( n = 3); (C) Uhrf1 knockdown weakened effects of EPT toward expressions of iNOS, COX‐2, IL‐1β, and pro IL‐1β in PKM2 overexpression cells; (D) COX‐2 and NLRP3 staining plots; (E) Uhrf1 knockdown led to the nuclear translocation of PKM2 in LPS‐mediated PKM2 overexpression cells; (F) Uhrf1 overexpression verification ( n = 3); (G) Uhrf1 overexpression enhanced effects of EPT toward mRNA levels of IL‐1β , iNOS , and COX‐2 in PKM2 overexpression cells ( n = 3); (H) Uhrf1 overexpression enhanced effects of EPT toward expressions of iNOS, COX‐2, IL‐1β, and pro IL‐1β in PKM2 overexpression cells; (I) COX‐2 and NLRP3 staining plots; (J) Uhrf1 overexpression suppressed the nuclear translocation of PKM2 in LPS‐mediated PKM2 overexpression cells.

Journal: Advanced Science

Article Title: Uhrf1‐Mediated PKM2 Degradation via Ubiquitination Alleviates Inflammation and Pyroptosis in Inflammatory Bowel Disease

doi: 10.1002/advs.77403

Figure Lengend Snippet: Anti‐inflammatory effects of EPT depended on Uhrf1‐mediated PKM2 ubiquitination. (A) Uhrf1 knockdown verification ( n = 3); (B) Uhrf1 knockdown weakened effects of EPT toward mRNA levels of IL‐1β , iNOS , and COX‐2 in PKM2 overexpression cells ( n = 3); (C) Uhrf1 knockdown weakened effects of EPT toward expressions of iNOS, COX‐2, IL‐1β, and pro IL‐1β in PKM2 overexpression cells; (D) COX‐2 and NLRP3 staining plots; (E) Uhrf1 knockdown led to the nuclear translocation of PKM2 in LPS‐mediated PKM2 overexpression cells; (F) Uhrf1 overexpression verification ( n = 3); (G) Uhrf1 overexpression enhanced effects of EPT toward mRNA levels of IL‐1β , iNOS , and COX‐2 in PKM2 overexpression cells ( n = 3); (H) Uhrf1 overexpression enhanced effects of EPT toward expressions of iNOS, COX‐2, IL‐1β, and pro IL‐1β in PKM2 overexpression cells; (I) COX‐2 and NLRP3 staining plots; (J) Uhrf1 overexpression suppressed the nuclear translocation of PKM2 in LPS‐mediated PKM2 overexpression cells.

Article Snippet: The primary antibodies occludin (27260‐1‐AP), RORγt (29910‐1‐AP), Foxp3 (22228‐1‐AP), GSDMD (66387‐1‐Ig), LDHA (19987‐1‐AP), GLUT1 (21829‐1‐AP), Mfn1 (13798‐1‐AP), Mfn2 (12186‐1‐AP), Opa1 (27733‐1‐AP), Fis1 (10956‐1‐AP), TOM20 (A19403), COX‐2 (66351‐1‐Ig), iNOS (22226‐1‐AP), p‐p65 (AP0124), p65 (10745‐1‐AP), caspase 1 (22915‐1‐AP), IL‐1β (26048‐1‐AP), ASC ( AB309497 ), NLRP3 (68102‐1‐Ig), PKM2 (60268‐1‐Ig), Drp1 (12957‐1‐AP), H3 (17168‐1‐AP), HA (51054‐1‐AP), GST (80006‐1‐AP), HIS (66005‐1‐AP), Ub (10201‐1‐AP), Uhrf1 (21402‐1‐AP), and GAPDH (60004‐1‐Ig) were purchased from Abcam (Shanghai, China), Abclonal (Wuhan, China) and Proteintech (Wuhan, China), respectively.

Techniques: Ubiquitin Proteomics, Knockdown, Over Expression, Staining, Translocation Assay