constipation mouse model  (MedChemExpress)

Journal: Research

Article Title: Inhibiting TRIM21 Neddylation Rejuvenates Oocyte Quality in PCOS by Regulating Ubiquitination of CPT1A

doi: 10.34133/research.1223

Figure Lengend Snippet: High expression of tripartite motif-containing protein 21 (TRIM21) in ovarian granulosa cells (GCs) of polycystic ovary syndrome (PCOS) mice. (A) Schematic illustration showing the induction of PCOS by prenatal anti-Müllerian hormone (PAMH) treatment. (B) Estrous cycle of adult female mice for 21 consecutive days ( n = 5). (C and D) Glucose tolerance test and insulin tolerance test after 16 h of fasting and 4 h of fasting in adult female mice ( n = 5). (E) Anogenital distance in adult female mice ( n = 15). (F) Number of pups per birth ( n = 10). (G and H) Testosterone and luteinizing hormone (LH) levels of serum ( n = 15). (I) Hematoxylin and eosin (H&E) staining of ovaries showing primordial follicles (*), growing follicles (#), and atretic follicles (arrows; scale bars, 100 μm). (J and K) Volcano plots showing differentially expressed proteins and genes in the ovaries of control and PAMH F 1 mice (up-regulated, red; down-regulated, blue; n = 3). (L) Up-regulated proteins and genes determined by proteomic and transcriptomic analysis. (M and N) Western blot (WB) and reverse transcription polymerase chain reaction (RT-PCR) analysis of TRIM21 in mouse ovarian tissues. (O and P) WB and RT-PCR analysis of TRIM21 in mouse GCs. (Q) Immunohistochemical staining of TRIM21 in mouse ovarian tissues (scale bars, 100 μm). Data are expressed as means ± SD, and each symbol represents a biologically independent mouse. Significance was calculated by 1-way analysis of variance (ANOVA) multiple comparison test. Blood glucose analysis between groups (C and D) was determined by 2-way ANOVA and multiple comparison test. ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: Sections were prepared and underwent immunohistochemical staining using the TRIM21 antibody (Cell Signaling Technology, cat# 92043) and CPT1A antibody (Cell Signaling Technology, cat# 97361), according to the standard procedures of the immunohistochemistry kit (KeyGEN, cat# KGC3201-300).

Techniques: Expressing, Staining, Control, Western Blot, Reverse Transcription, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Immunohistochemical staining, Comparison

Journal: Research

Article Title: Inhibiting TRIM21 Neddylation Rejuvenates Oocyte Quality in PCOS by Regulating Ubiquitination of CPT1A

doi: 10.34133/research.1223

Figure Lengend Snippet: Tripartite motif-containing protein 21 (TRIM21) is associated with fatty acid oxidation (FAO) in ovarian granulosa cells. (A) Volcano plot for the significant ( P < 0.05) alterations in protein expression induced by TRIM21 knockdown ( n = 3). (B) Heatmap plot showing differentially expressed proteins ( n = 3). (C) Gene Ontology (GO) enrichment analysis of the proteomic sequencing. (D and E) Evaluation of the mitochondrial oxidative phosphorylation (OXPHOS) function by oxygen consumption rate (OCR) in KGN cells, including basal respiration, maximum respiration, adenosine triphosphate (ATP) production, and coupling efficiency ( n = 6). (F) Evaluation of the glycolysis function by extracellular acidification rate (ECAR) in KGN cells ( n = 3). (G and H) Evaluation of FAO-dependent mitochondrial function by OCR in KGN cells, including basal respiration and maximum respiration ( n = 6). (I) transmission electron microscopy (TEM) analysis of mitochondrial morphology in KGN cell. Green arrows indicate normal mitochondria, and red arrows indicate abnormal mitochondria ( n = 5; scale bars, 2 μm). (J) Representative images for JC-1 ( n = 15; scale bar, 50 μm) and ratio of the fluorescence intensities in KGN cells. (K) Representative images for tetramethylrhodamine ethyl ester perchlorate (TMRE) ( n = 15; scale bar, 50 μm) and ratio of the fluorescence intensities in KGN cells. (L) Activity of mitochondrial complexes I to V in KGN cells ( n = 6). (M) Western blot (WB) analysis of OXPHOS subunits (complex I-NDUFB8, complex II-SDHB, complex III-UQCRC2, complex IV-MTCO1, and complex V-ATP5A1) of KGN cells. Data are expressed as means ± standard error of the mean (SEM). ns, not significant; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: Sections were prepared and underwent immunohistochemical staining using the TRIM21 antibody (Cell Signaling Technology, cat# 92043) and CPT1A antibody (Cell Signaling Technology, cat# 97361), according to the standard procedures of the immunohistochemistry kit (KeyGEN, cat# KGC3201-300).

Techniques: Expressing, Knockdown, Sequencing, Phospho-proteomics, Transmission Assay, Electron Microscopy, Fluorescence, Activity Assay, Western Blot

Journal: Research

Article Title: Inhibiting TRIM21 Neddylation Rejuvenates Oocyte Quality in PCOS by Regulating Ubiquitination of CPT1A

doi: 10.34133/research.1223

Figure Lengend Snippet: Tripartite motif-containing protein 21 (TRIM21) regulates fatty acid oxidation in granulosa cells through carnitine palmitoyltransferase 1A (CPT1A). (A) CPT1A was identified as a protein with high confidence interacting with TRIM21 by immunoprecipitation-mass spectrometry (IP-MS) and proteomic sequencing. (B) Western blot (WB) analysis of KGN cell lysates immunoprecipitated with TRIM21 or CPT1A antibodies. (C and D) Coimmunoprecipitation (Co-IP) and WB analysis of exogenous TRIM21 and CPT1A in KGN cells overexpressing Myc-TRIM21 and Flag- CPT1A. (E) Immunofluorescence staining ofTRIM21 and CPT1A by confocal microscopy (scale bar, 10 μm) in KGN cells. (F) Molecular docking of TRIM21 and CPT1A. (G) Microscale thermophoresis of TRIM21 and CPT1A. (H to J) WB analysis of CPT1A with TRIM21 silencing or overexpression in KGN cells. (K) WB analysis of ATP5A1 with CPT1A silencing, with or without TRIM21 knockdown in KGN cells. (L) WB analysis of ATP5A1 in cells with etomoxir, with or without TRIM21 knockdown in KGN cells. (M and N) Evaluation of the mitochondrial oxidative phosphorylation (OXPHOS) function by oxygen consumption rate (OCR), including basal respiration and maximum respiration ( n = 6). (O) Representative images for tetramethylrhodamine ethyl ester perchlorate (TMRE) in oocytes ( n = 15; scale bar, 50 μm) and relative fluorescence intensities. Three fields of view of each mouse were selected. (P) Representative images for lipid content in oocytes by ( n = 15; scale bar, 50 μm) and relative fluorescence intensities. Three fields of view of each mouse were selected. Data are expressed as means ± standard error of the mean (SEM). ns, not significant; **** P < 0.0001. IB, immunoblot.

Article Snippet: Sections were prepared and underwent immunohistochemical staining using the TRIM21 antibody (Cell Signaling Technology, cat# 92043) and CPT1A antibody (Cell Signaling Technology, cat# 97361), according to the standard procedures of the immunohistochemistry kit (KeyGEN, cat# KGC3201-300).

Techniques: Immunoprecipitation, Mass Spectrometry, Protein-Protein interactions, Sequencing, Western Blot, Co-Immunoprecipitation Assay, Immunofluorescence, Staining, Confocal Microscopy, Microscale Thermophoresis, Over Expression, Knockdown, Phospho-proteomics, Fluorescence

Journal: Research

Article Title: Inhibiting TRIM21 Neddylation Rejuvenates Oocyte Quality in PCOS by Regulating Ubiquitination of CPT1A

doi: 10.34133/research.1223

Figure Lengend Snippet: Tripartite motif-containing protein 21 (TRIM21) facilitates ubiquitination of carnitine palmitoyltransferase 1A (CPT1A) at lysine 161. (A) Reverse transcription polymerase chain reaction (RT-PCR) analysis of CPT1A with or without TRIM21 knockdown of KGN and COV434 cells ( n = 5). (B) Western blot (WB) analysis of CPT1A with TRIM21 overexpression in the presence of MG132 in KGN cells. (C) WB analysis of CPT1A in KGN cells treated with cycloheximide (CHX) and MG132 at different time points. (D) WB analysis of CPT1A in cells with or without TRIM21 silencing in CHX-chase experiment in KGN cells. (E and F) In KGN cells, coimmunoprecipitation (Co-IP) and WB analysis of CPT1A exogenous ubiquitination with TRIM21 knockdown or overexpression in the presence of MG132 (10 μM, 4 h). (G and H) In KGN cells, Co-IP and WB analysis of CPT1A endogenous ubiquitination with TRIM21 knockdown or overexpression in the presence of MG132 (10 μM, 4 h). (I and J) In KGN cells, Co-IP and WB analysis of exogenous ubiquitination of CPT1A treated with Flag-CPT1A and HA-UB (wild type [WT], K48-only, and K63-only) in the presence of MG132 (10 μM, 4 h). (K and L) Analysis of ubiquitination modification sites of CPT1A by immunoprecipitation-mass spectrometry (IP-MS). (M and N) In KGN cells, Co-IP and WB of exogenous ubiquitination of CPT1A treated with Flag-CPT1A mutant plasmids in the presence of MG132 (10 μM, 4 h). Data are expressed as means ± SD. IB, immunoblot.

Article Snippet: Sections were prepared and underwent immunohistochemical staining using the TRIM21 antibody (Cell Signaling Technology, cat# 92043) and CPT1A antibody (Cell Signaling Technology, cat# 97361), according to the standard procedures of the immunohistochemistry kit (KeyGEN, cat# KGC3201-300).

Techniques: Ubiquitin Proteomics, Reverse Transcription, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Knockdown, Western Blot, Over Expression, Co-Immunoprecipitation Assay, Modification, Immunoprecipitation, Mass Spectrometry, Protein-Protein interactions, Mutagenesis

Journal: Research

Article Title: Inhibiting TRIM21 Neddylation Rejuvenates Oocyte Quality in PCOS by Regulating Ubiquitination of CPT1A

doi: 10.34133/research.1223

Figure Lengend Snippet: UBE2M promotes tripartite motif-containing protein 21 (TRIM21) neddylation and its interaction with carnitine palmitoyltransferase 1A (CPT1A). (A) UBE2M was identified as a protein with high confidence interacting with TRIM21 by immunoprecipitation-mass spectrometry (IP-MS) and functional analysis. (B) Molecular docking of UBE2M and TRIM21. (C and D) Coimmunoprecipitation (Co-IP) and Western blot (WB) analysis of exogenous UBE2M and TRIM21 in KGN cells overexpressing Flag-UBE2M and MYC-TRIM21. (E) Immunofluorescence staining of UBE2M and TRIM21 by confocal microscopy in KGN cells (scale bar, 20 μm). (F) WB analysis of TRIM21 and neural precursor cell-expressed developmentally down-regulated 8 (NEDD8) with UBE2M knockdown in KGN cells. (G) WB analysis of KGN cell lysates immunoprecipitated with immunoglobulin G (IgG) and anti-NEDD8 antibodies. (H) Immunofluorescence staining of TRIM21 and NEDD8 by confocal microscopy in KGN cells (scale bar, 20 μm). (I) WB analysis of TRIM21 in KGN cells overexpressing UBE2M with or without NEDD8 knockdown. (J) Co-IP and WB analysis of TRIM21 neddylation with or without UBE2M knockdown in KGN cells. (K) WB analysis of CPT1A in KGN cells treated with different concentrations of FLAG-UBE2M. (L) WB analysis of CPT1A treated with UBE2M silencing, with or without TRIM21 knockdown in KGN cells. (M) Co-IP and WB analysis of KGN cell lysates immunoprecipitated with anti-MYC antibody in the presence of UBE2M knockdown. (N) In KGN cells, Co-IP and WB analysis of TRIM21 neddylation, CPT1A and ATP5A1 with UBE2M silencing, with or without NEDD8 knockdown. (O) In KGN cells, WB analysis of K48 ubiquitination of CPT1A in cells with UBE2M overexpression, with or without TRIM21 knockdown in the presence of MG132 (10 μM, 4 h) treatment. (P) In KGN cells, Co-IP and WB analysis of exogenous K48 ubiquitination of CPT1A in KGN cells with UBE2M overexpression, with or without NEDD8 knockdown in the presence of MG132 (10 μM, 4 h). IB, immunoblot.

Article Snippet: Sections were prepared and underwent immunohistochemical staining using the TRIM21 antibody (Cell Signaling Technology, cat# 92043) and CPT1A antibody (Cell Signaling Technology, cat# 97361), according to the standard procedures of the immunohistochemistry kit (KeyGEN, cat# KGC3201-300).

Techniques: Immunoprecipitation, Mass Spectrometry, Protein-Protein interactions, Functional Assay, Co-Immunoprecipitation Assay, Western Blot, Immunofluorescence, Staining, Confocal Microscopy, Knockdown, Ubiquitin Proteomics, Over Expression

Journal: Research

Article Title: Inhibiting TRIM21 Neddylation Rejuvenates Oocyte Quality in PCOS by Regulating Ubiquitination of CPT1A

doi: 10.34133/research.1223

Figure Lengend Snippet: MLN4924 reverses the ubiquitination of carnitine palmitoyltransferase 1A (CPT1A) by tripartite motif-containing protein 21 (TRIM21) and ameliorates the phenotype of polycystic ovary syndrome (PCOS) mice. (A) Western blot (WB) analysis of neural precursor cell-expressed developmentally down-regulated 8 (NEDD8) and CPT1A treated with MLN4924, with or without TRIM21 knockdown in KGN cell. (B and C) Coimmunoprecipitation (Co-IP) and WB analysis of TRIM21 neddylation in KGN cells treated with or without MLN4924. (D) Co-IP and WB analysis of exogenous K48 ubiquitination of CPT1A in KGN cells overexpressing UBE2M, with or without NEDD8 knockdown in the presence of MG132 (10 μM, 4 h). (E and F) Evaluation of the mitochondrial oxidative phosphorylation (OXPHOS) function by oxygen consumption rate (OCR) in KGN cells, including basal respiration, maximum respiration, ATP generation, and coupling efficiency ( n = 6). (G and H) Evaluation of fatty acid oxidation (FAO)-dependent mitochondrial function by OCR in KGN cells, including basal respiration and maximum respiration ( n = 6). (I) Activity of mitochondrial complex V in KGN cells ( n = 6). (J and K) Testosterone and luteinizing hormone (LH) levels of serum ( n = 15). (L) Anogenital distance in adult female mice ( n = 15). (M) Insulin tolerance test (ITT) test in adult female mice after 4 h of fasting ( n = 5). (N) Number of pups per birth ( n = 10). (O and P) WB and reverse transcription polymerase chain reaction (RT-PCR) analysis of TRIM21 and CPT1A in ovarian granulosa cells (GCs). (Q) Immunohistochemical staining of TRIM21 in mouse ovarian tissues (scale bars, 100 μm). Data are expressed as means ± standard error of the mean (SEM), and each symbol represents a biologically independent mouse. Significance was calculated by 1-way analysis of variance (ANOVA) multiple comparison test. Blood glucose analysis between groups (M) was determined by 2-way ANOVA and multiple comparison test. ns, not significant; ** P < 0.01; *** P < 0.001; **** P < 0.0001. IB, immunoblot.

Article Snippet: Sections were prepared and underwent immunohistochemical staining using the TRIM21 antibody (Cell Signaling Technology, cat# 92043) and CPT1A antibody (Cell Signaling Technology, cat# 97361), according to the standard procedures of the immunohistochemistry kit (KeyGEN, cat# KGC3201-300).

Techniques: Ubiquitin Proteomics, Western Blot, Knockdown, Co-Immunoprecipitation Assay, Phospho-proteomics, Activity Assay, Reverse Transcription, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Immunohistochemical staining, Staining, Comparison

Journal: Research

Article Title: Inhibiting TRIM21 Neddylation Rejuvenates Oocyte Quality in PCOS by Regulating Ubiquitination of CPT1A

doi: 10.34133/research.1223

Figure Lengend Snippet: MLN4924 improves the quality of oocytes of polycystic ovary syndrome (PCOS) mice. (A) Hematoxylin and eosin (H&E) staining of ovaries and the number of follicles in ovary including primordial follicles (*), growing follicles (#), and atretic follicles (arrows; scale bars, 100 μm). (B) Number of oocytes after superovulation ( n = 5 mice; scale bars, 100 μm). (C) Representative images and percentage of first polar body extrusion in mouse oocytes, fertilization rate, and blastocyst maturation rate ( n = 5 mice; scale bars, 100 μm). (D) Transmission electron microscopy (TEM) analysis of mitochondrial morphology of mouse ovarian granulosa cells. Green arrows indicate normal mitochondria, red arrows indicate abnormal mitochondria, and # indicates lipid droplets ( n = 5 mice; scale bar, 500 nm). (E) Representative images of spindle morphology and chromosome alignment in oocytes at the metaphase II stage by confocal microscopy ( n = 5 mice; scale bar, 10 μm). The percentage of aberrant spindles and misaligned chromosomes were quantified in oocytes at metaphase II from control ( n = 23), PCOS ( n = 19), and MLN4924 ( n = 21) mice. (F) Representative images for JC-1 staining of mouse oocytes ( n = 15; scale bar, 50 μm) and relative fluorescence intensities. Three fields of view of each mouse were selected (G) Representative images for tetramethylrhodamine ethyl ester perchlorate (TMRE) staining of mouse oocytes ( n = 15; scale bar, 50 μm) and relative fluorescence intensities. Three fields of view of each mouse were selected. (H) Representative images for lipid content in oocytes ( n = 15; scale bar, 50 μm) and ratio of the fluorescence intensities. Three fields of view of each mouse were selected. (I) Reverse transcription polymerase chain reaction (RT-PCR) analysis of mRNA levels of follicle development-related genes in mouse cumulus–oocyte complexes (COCs) ( n = 5). Data are expressed as means ± SD, and each symbol represents a biologically independent mouse. (J) Schematic diagram of the pathway by which tripartite motif-containing protein 21 (TRIM21) regulates ubiquitination of carnitine palmitoyltransferase 1A (CPT1A) leading to abnormal fatty acid oxidation in ovarian granulosa cells. ns, not significant; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: Sections were prepared and underwent immunohistochemical staining using the TRIM21 antibody (Cell Signaling Technology, cat# 92043) and CPT1A antibody (Cell Signaling Technology, cat# 97361), according to the standard procedures of the immunohistochemistry kit (KeyGEN, cat# KGC3201-300).

Techniques: Staining, Transmission Assay, Electron Microscopy, Confocal Microscopy, Control, Fluorescence, Reverse Transcription, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Ubiquitin Proteomics

Journal: Journal of Biomedical Science

Article Title: IFI35 suppresses the transcription of hepatitis B virus cccDNA minichromosome via promoting HNF4α proteasomal degradation

doi: 10.1186/s12929-026-01239-w

Figure Lengend Snippet: IFI35 ubiquitinates HNF4a in TRIM21-dependent manner. A Cell lysates were immunoprecipitated using an anti-TRIM21 antibody. B Detection of HBV DNAs by Southern blot from HepG2 and HepG2-TRIM21-KO cells. Expression levels of HNF4α, IFI35 and TRIM21 in HepG2-TRIM21-KO cells compared to parental HepG2 cells (Control) were assessed by Western blot. HBV DNA levels of HepG2 and HepG2-TRIM21-KO cells were quantified in graph (bottom). C Quantification of HNF4α protein levels in HepG2 and HepG2-TRIM21 knockout (KO) cells transfected with increasing amounts of myc-IFI35. HNF4α levels were determined by immunoblotting and quantified relative to the vector control. Data represent mean ± SD from three independent experiments. D Secreted HBeAg and HBsAg levels were measured by ELISA in HepG2 and HepG2-TRIM21-KO cells transfected with increasing amounts of myc-IFI35. Data represent mean ± SD from three independent experiments. E HepG2 or HepG2-TRIM21 knockout cells were transfected with HBV 1.2mer together with increasing amounts of IFI35. Total RNA was analyzed by Northern blotting to detect HBV transcription. F Effect of TRIM21 on IFI35-mediated HNF4α degradation. HepG2 and HepG2-TRIM21-KO cells were transfected with either empty vector or myc-IFI35. After 48 h, cycloheximide (50ug/ml) were treated for the indicated times, and cells were harvested. Expression levels of HNF4α and IFI35 were evaluated through Western blotting. Quantification of HNF4α levels between HepG2 and HepG2-TRIM21-KO cells (bottom). G HepG2 cells and TRIM21 knockout cells were co-transfected with HA-tagged ubiquitin (1 μg) and either vector or IFI35 (2 μg). At 48 h post-transfection, HNF4α was immunoprecipitated and immunoblotted with anti-ubiquitin antibody to assess polyubiquitination. Total levels of HNF4α, IFI35, TRIM21, and HA-tagged ubiquitin in input lysates were also analyzed by immunoblotting. H Schematic diagram of HBV enhancer luciferase reporter constructs. Constructs containing enhancer I (EnhI), enhancer II (EnhII), or both regions were cloned upstream of the luciferase reporter gene. Mutant constructs lacking HNF4α binding sites were generated in EnhI (ΔHNF4α) or EnhII (ΔHNF4α1 and ΔHNF4α1,2) as indicated. I Basal luciferase activity of HBV enhancer reporter constructs. HepG2 cells were transfected with the indicated luciferase reporter plasmids, and luciferase activity was measured. J Comparison of the effect of IFI35 on EnhI-driven transcription with or without the HNF4α binding site. HepG2 cells were co-transfected with the indicated EnhI luciferase reporters and increasing amounts of myc-IFI35. Luciferase activity was measured 48 h post-transfection. Data represent mean ± SD from three independent experiments. K Comparison of the effect of IFI35 on EnhII-driven transcription with or without HNF4α binding sites. HepG2 cells were co-transfected with EnhII luciferase reporters containing wild-type or mutated HNF4α binding sites together with increasing amounts of myc-IFI35. Luciferase activity was measured 48 h post-transfection. Data represent mean ± SD from three independent experiments. The above experiment was independently conducted three times. Data are shown as mean ± SD. The statistical significance of the differences was assessed by the Student t test: *, P < 0.05; **, P < 0.01, ***, P < 0.001

Article Snippet: The following antibodies were used: IFI35 (sc-100769, Santa Cruz), HNF4α (H-171, Santa Cruz), HNF1α (Η-140, Santa Cruz), HNF3β (RY-7, Santa Cruz), core protein (B0586, Dako, Carpinteria, CA), TRIM21 (sc-25351, Santa Cruz), β-actin (A5441, Sigma) and GFP (sc-9996, Santa Cruz).

Techniques: Immunoprecipitation, Southern Blot, Expressing, Control, Western Blot, Knock-Out, Transfection, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Northern Blot, Ubiquitin Proteomics, Luciferase, Construct, Clone Assay, Mutagenesis, Binding Assay, Generated, Activity Assay, Comparison

Journal: Journal of Biomedical Science

Article Title: IFI35 suppresses the transcription of hepatitis B virus cccDNA minichromosome via promoting HNF4α proteasomal degradation

doi: 10.1186/s12929-026-01239-w

Figure Lengend Snippet: Domain mapping of IFI35 as mediators of TRIM21–HNF4α complex formation. A Schematic representation of IFI35 constructs used in this study. GFP-tagged mutants lacking NID1 (ΔNID1) or NID2 (ΔNID2), as well as the leucine zipper construct (Zip), were generated. B HepG2 cells were co-transfected with HBV 1.2mer and the indicated IFI35 constructs. At 72 h post-transfection, HBV replication intermediates were analyzed by Southern blotting. Quantification of HBV replication levels based on Southern blot analysis. C Secreted HBeAg and HBsAg levels in the culture supernatants were measured by ELISA. E Co-immunoprecipitation analysis of IFI35 mutants with HNF4α. HepG2 cells were transfected with GFP-tagged IFI35 constructs and cell lysates were immunoprecipitated using anti-HNF4α antibody followed by immunoblotting with anti-GFP antibody. F Interaction of IFI35 mutants with TRIM21. Cells were transfected with Myc-TRIM21 together with the indicated IFI35 constructs. Cell lysates were subjected to immunoprecipitation using anti-TRIM21 antibody followed by immunoblotting with anti-GFP antibody. Data represent mean ± SD (n = 3). Statistical significance was determined relative to the vector control

Article Snippet: The following antibodies were used: IFI35 (sc-100769, Santa Cruz), HNF4α (H-171, Santa Cruz), HNF1α (Η-140, Santa Cruz), HNF3β (RY-7, Santa Cruz), core protein (B0586, Dako, Carpinteria, CA), TRIM21 (sc-25351, Santa Cruz), β-actin (A5441, Sigma) and GFP (sc-9996, Santa Cruz).

Techniques: Construct, Generated, Transfection, Southern Blot, Enzyme-linked Immunosorbent Assay, Immunoprecipitation, Western Blot, Plasmid Preparation, Control

Journal: Research

Article Title: TRIM21 Exacerbates Ischemic Brain Injury by Promoting Astrocyte-Mediated Neuroinflammation via K63-Linked Ubiquitination of MDA5

doi: 10.34133/research.1200

Figure Lengend Snippet: TRIM21 deficiency reduces brain ischemic volume and improves neurological recovery after tMCAO.​ (A) Volcano plots of differentially expressed genes from GSE112348 and GSE202391 datasets. (B) A Venn diagram highlighted 5 overlapping TRIM family genes, including TRIM21. (C) Western blot images and quantification of TRIM21 protein expression in ischemic brain tissue from WT mice at 6, 12, 24, and 72 h post-tMCAO ( n = 5, Welch’s ANOVA followed by Dunnett’s T3 post hoc test). (D) Schematic diagram of experimental design for (E) and (F). (E) Representative T2-weighted MRI images and quantification of infarct volume in WT and TRIM21 −/− mice on day 3 post-tMCAO ( n = 8, unpaired t test). (F) Behavioral tests, including mNSS score, rotarod test, wire-hanging test, and adhesive removal test, were assessed before the surgery and on days 1, 3, 5, and 7 post-tMCAO ( n = 9 to 15, 2-way ANOVA followed by Tukey’s post hoc test). All data are expressed as means ± SD. * P < 0.05 and *** P < 0.001 versus indicated groups; # P < 0.05, ## P < 0.01, and ### P < 0.001 versus tMCAO + TRIM21 −/− group.

Article Snippet: WT mice and Trim21 KO mice (NM-KO-190403) were obtained from Shanghai Model Organisms Center.

Techniques: Western Blot, Expressing, Adhesive

Journal: Research

Article Title: TRIM21 Exacerbates Ischemic Brain Injury by Promoting Astrocyte-Mediated Neuroinflammation via K63-Linked Ubiquitination of MDA5

doi: 10.34133/research.1200

Figure Lengend Snippet: TRIM21 deficiency alleviates BBB damage and neuronal injury after tMCAO.​ (A and B) Western blot images and quantification of ZO-1 and Occludin protein expression in ischemic brain tissue of WT and TRIM21 −/− mice at 3 d post-tMCAO ( n = 5, one-way ANOVA followed by Tukey’s post hoc test). (C and D) Representative images and quantitative analysis of the Evans Blue extravasation in the brain tissue of WT and TRIM21 −/− mice after tMCAO ( n = 5, Welch’s ANOVA followed by Dunnett’s T3 post hoc test). (E) TUNEL staining demonstrating cell death in the peri-infarct hippocampus and cortex of WT and TRIM21 −/− mice. Scale bar, 50 μm. (F and G) Quantitative analysis of TUNEL-positive cells in the hippocampus (F) and cortex (G) ( n = 5, Welch’s ANOVA followed by Dunnett’s T3 post hoc test). All data are expressed as means ± SD. * P < 0.05, ** P < 0.01, and *** P < 0.001 versus indicated groups.

Article Snippet: WT mice and Trim21 KO mice (NM-KO-190403) were obtained from Shanghai Model Organisms Center.

Techniques: Western Blot, Expressing, TUNEL Assay, Staining

Journal: Research

Article Title: TRIM21 Exacerbates Ischemic Brain Injury by Promoting Astrocyte-Mediated Neuroinflammation via K63-Linked Ubiquitination of MDA5

doi: 10.34133/research.1200

Figure Lengend Snippet: TRIM21 expression is up-regulated in astrocytes of the brain after ischemic stroke. (A to C) Immunofluorescence of TRIM21 in the peri-infarct region of the ischemic mice at 72 h post-tMCAO. Double immunofluorescence of TRIM21 (green) and GFAP (astrocyte marker, red) (A), Iba1 (microglia marker, red) (B), and NeuN (neuron marker, red) (C) was performed. Scale bars, 20 μm. (D) Western blot images and quantification of TRIM21 in primary astrocytes subjected to 6-h OGD, followed by 3, 6, 12, and 24 h of reoxygenation ( n = 5, Welch’s ANOVA followed by Dunnett’s T3 post hoc test). (E) Western blot images and quantification of TRIM21 in primary neurons and microglia subjected to OGD (2 h for neurons and 3 h for microglia), followed by 12- and 24-h reoxygenation ( n = 5, one-way ANOVA followed by Tukey’s post hoc test). (F) Representative immunofluorescence images of TRIM21 in primary astrocytes. Double immunofluorescence of TRIM21 (green) and GFAP (astrocyte marker, red) was performed. Scale bars, 20 μm. All data are expressed as means ± SD. ** P < 0.01 versus indicated groups.

Article Snippet: WT mice and Trim21 KO mice (NM-KO-190403) were obtained from Shanghai Model Organisms Center.

Techniques: Expressing, Immunofluorescence, Marker, Western Blot

Journal: Research

Article Title: TRIM21 Exacerbates Ischemic Brain Injury by Promoting Astrocyte-Mediated Neuroinflammation via K63-Linked Ubiquitination of MDA5

doi: 10.34133/research.1200

Figure Lengend Snippet: TRIM21 deficiency attenuates inflammation and oxidative stress following cerebral I/R injury in vivo and in vitro. (A to C) RNA-sequencing analysis in the ischemic hemispheres of WT and TRIM21 −/− mice at 72 h post-tMCAO ( n = 3 mice per group). Heatmaps of gene expression (A), GO enrichment analysis (B), and Gene Set Enrichment Analysis (GSEA) (C) of tMCAO-TRIM21 −/− groups and tMCAO-WT groups. (D) Schematic diagram of experimental design for (E) and (F). (E and F) The ischemic hemispheres of WT and TRIM21 −/− mice at 72 h post-tMCAO were collected and homogenized to determine the levels of proinflammatory cytokines (IL-1β, IL-6, and TNF-α) (E) and oxidative stress (MDA, SOD, and GSH-Px) (F). n = 5. (G) Schematic diagram of experimental design for (H) and (I). (H) ELISA analysis of IL-1β, IL-6, and TNF-α levels in the culture medium of OGD/R-treated WT and TRIM21 −/− primary astrocytes. n = 5. (I) Effects of TRIM21 on MDA, SOD, and GSH-Px in OGD/R-treated primary astrocytes. n = 5. (J) Representative fluorescence staining images and quantitative analysis of ROS levels. Scale bars, 100 μm. n = 5. All data are expressed as means ± SD. In (E), IL-1β and TNF-α were analyzed by Welch’s ANOVA with Dunnett’s T3 test, while IL-6 was analyzed by one-way ANOVA with Tukey’s post hoc test. In (F), MDA was analyzed by Welch’s ANOVA with Dunnett’s T3 test, while SOD and GSH-Px were analyzed by one-way ANOVA with Tukey’s post hoc test. In (H) and (I), data were analyzed by one-way ANOVA with Tukey’s post hoc test. In (J), data were analyzed by Welch’s ANOVA with Dunnett’s T3 test. * P < 0.05, ** P < 0.01, and *** P < 0.001 versus indicated groups.

Article Snippet: WT mice and Trim21 KO mice (NM-KO-190403) were obtained from Shanghai Model Organisms Center.

Techniques: In Vivo, In Vitro, RNA Sequencing, Gene Expression, Enzyme-linked Immunosorbent Assay, Fluorescence, Staining

Journal: Research

Article Title: TRIM21 Exacerbates Ischemic Brain Injury by Promoting Astrocyte-Mediated Neuroinflammation via K63-Linked Ubiquitination of MDA5

doi: 10.34133/research.1200

Figure Lengend Snippet: MDA5 is a potential interaction partner for TRIM21. (A) The schematic diagram showed that LC-MS/MS analysis was used to identify proteins interacting with TRIM21. (B) Gene Ontology (GO) enrichment analysis of potential interaction partners for TRIM21 in vivo and in vitro models. (C) The Venn diagram illustrated the proteins identified in both the LC-MS/MS databases and the Ubi-Browser dataset. (D) Protein–protein docking visualization (green for MDA5, blue for TRIM21). (a) Docking interaction surfaces of TRIM21 and MDA5 (indicated in purple). (b) Microscopic diagram showing the mechanism of TRIM21–MDA5 interaction. (E) Western blot images and quantification of MDA5 protein expression in ischemic brain tissue from WT mice at 6, 12, 24, and 72 h post-tMCAO ( n = 5, Welch’s ANOVA followed by Dunnett’s T3 post hoc test). (F) Immunofluorescence of MDA5 in the peri-infarct region of the ischemic mice at 72 h post-tMCAO. Double immunofluorescence of MDA5 (green) and GFAP (astrocyte marker, red) was performed. Scale bars, 20 μm. (G) Western blot images and quantification of MDA5 in primary astrocytes subjected to 6-h OGD, followed by 3, 6, 12, and 24 h of reoxygenation ( n = 5, Welch’s ANOVA followed by Dunnett’s T3 post hoc test). (H) Representative immunofluorescence image of MDA5 in primary astrocytes. Double immunofluorescence of MDA5 (green) and GFAP (astrocyte marker, red) was performed. Scale bars, 20 μm. All data are expressed as means ± SD. ** P < 0.01, *** P < 0.001, and **** P < 0.0001 versus indicated groups.

Article Snippet: WT mice and Trim21 KO mice (NM-KO-190403) were obtained from Shanghai Model Organisms Center.

Techniques: Liquid Chromatography with Mass Spectroscopy, In Vivo, In Vitro, Western Blot, Expressing, Immunofluorescence, Marker

Journal: Research

Article Title: TRIM21 Exacerbates Ischemic Brain Injury by Promoting Astrocyte-Mediated Neuroinflammation via K63-Linked Ubiquitination of MDA5

doi: 10.34133/research.1200

Figure Lengend Snippet: TRIM21 interacts with MDA5. (A) IP analysis of the endogenous interaction of TRIM21 with MDA5 in primary astrocytes. (B) Representative images of laser scanning confocal microscopy for TRIM21 (red) and MDA5 (green) in primary astrocytes subjected to OGD/R. The following 2 panels indicate the line profiling of TRIM21 with MDA5 under control and OGD/R stimulation conditions, respectively, and the intensity of each line was quantified by ImageJ software and drawn by GraphPad Prism 10.0. Scale bars, 20 μm. (C) IP analysis of the exogenous interaction of TRIM21 with MDA5 in HEK293T cells transfected with plasmids expressing HA-TRIM21 and Flag-MDA5. (D) Representative images of laser scanning confocal microscopy for HA (red) and Flag (green) in HEK293T cells. The following panel indicates the line profiling of exogenous TRIM21 with MDA5, and the intensity of each line was quantified by ImageJ software and drawn by GraphPad Prism 10.0. Scale bars, 10 μm. (E) Schematic diagram of TRIM21 and its truncation mutants. (F) HA-tagged TRIM21 or its mutants and Flag-MDA5 were individually transfected into HEK293T cells. The cell lysates were immunoprecipitated with an anti-HA antibody and then immunoblotted with the indicated antibody. (G) Schematic diagram of MDA5 and its truncation mutants. (H) Flag-tagged MDA5 or its mutants and HA-TRIM21 were individually transfected into HEK293T cells. The cell lysates were immunoprecipitated with an anti-Flag antibody and then immunoblotted with the indicated antibody. (I) Surface plasmon resonance (SPR) analysis for direct TRIM21–MDA5 interaction. Results are representative of 3 independent experiments.

Article Snippet: WT mice and Trim21 KO mice (NM-KO-190403) were obtained from Shanghai Model Organisms Center.

Techniques: Confocal Microscopy, Control, Software, Transfection, Expressing, Immunoprecipitation, SPR Assay

Journal: Research

Article Title: TRIM21 Exacerbates Ischemic Brain Injury by Promoting Astrocyte-Mediated Neuroinflammation via K63-Linked Ubiquitination of MDA5

doi: 10.34133/research.1200

Figure Lengend Snippet: TRIM21 mediates K63-linked polyubiquitination of MDA5 to enhance NF-κB signaling and exacerbates ischemic brain injuries. (A) IP analysis of the ubiquitination of MDA5 in HEK293T cells transfected with plasmids expressing Flag-MDA5, Myc-ubiquitin (Myc-Ub), and HA-TRIM21 (WT or C31A, H33W). (B) HEK293T cells were transfected with plasmids expressing Myc-Ub (WT or K48, K63), HA-TRIM21, and Flag-MDA5. IP analysis of the polyubiquitination forms of MDA5 mediated by TRIM21. (C) Endogenous ubiquitination levels of MDA5 in WT and TRIM21 −/− primary astrocytes were examined by IP and Western blot. (D) Half-life of MDA5 was measured by the CHX assay in WT and TRIM21 −/− primary astrocytes ( n = 3, 2-way ANOVA followed by Tukey’s post hoc test). (E) Western blot images and quantification of phosphorylation level of TBK1 and P65 in WT and TRIM21 −/− primary astrocytes after OGD/R ( n = 5, Welch’s ANOVA followed by Dunnett’s T3 post hoc test). (F) Schematic diagram of experimental design for (G) to (J). (G) Representative immunofluorescence images of GFAP (red) and MDA5 (green) in AAV-GfaABC1D-MDA5-infected mice on day 21 after injection. Scale bars, 20 μm. (H) Representative T2-weighted MRI images and quantification of infarct volume in AAV-infected TRIM21 −/− mice on day 3 post-tMCAO ( n = 8, unpaired t test). (I) The mNSS test was assessed in AAV-infected TRIM21 −/− mice on day 3 post-tMCAO ( n = 11, unpaired t test). (J) RT-qPCR analysis of proinflammatory cytokine mRNA levels in ischemic hemispheres of AAV-infected TRIM21 −/− mice on day 3 post-tMCAO ( n = 5, Welch’s t test). All data are expressed as means ± SD. * P < 0.05, ** P < 0.01, and *** P < 0.001 versus indicated groups.

Article Snippet: WT mice and Trim21 KO mice (NM-KO-190403) were obtained from Shanghai Model Organisms Center.

Techniques: Ubiquitin Proteomics, Transfection, Expressing, Western Blot, Phospho-proteomics, Immunofluorescence, Infection, Injection, Quantitative RT-PCR

Journal: Research

Article Title: TRIM21 Exacerbates Ischemic Brain Injury by Promoting Astrocyte-Mediated Neuroinflammation via K63-Linked Ubiquitination of MDA5

doi: 10.34133/research.1200

Figure Lengend Snippet: Engineered nanoparticle-mediated delivery of targeted TRIM21 silencing promotes neurological recovery after cerebral I/R. (A) Schematic illustration of the preparation of siTRIM21@RVG-PLGA NPs. (B) Evaluation of siTRIM21@PLGA NPs and siTRIM21@RVG-PLGA NPs detected by dynamic light scattering. (C) Transmission electron microscopy image of siTRIM21@RVG-PLGA NPs. Scale bars, 100 nm. (D) Pseudo-color images showed the DiR fluorescence of the in vivo brain 4, 8, 12, and 24 h after injection, and the scatterplot showed radiant efficiency ( n = 5, 2-way ANOVA followed by Tukey’s post hoc test). (E) Pseudo-color images showed the DiR fluorescence of the ex vivo brain 24 h after injection. (F) Representative images of GFAP staining (red) in brain sections derived from mice treated with siTRIM21@RVG-PLGA NPs-DiO (green) at 24 h post-injection. (G) Schematic diagram of experimental design for (H) and (I). (H) Representative T2-weighted MRI images and quantification of infarct volume in the mice treated with siTRIM21@RVG-PLGA NPs or siNC@RVG-PLGA NPs on day 3 post-tMCAO ( n = 8, unpaired t test). (I) Behavioral test results of the mNSS score and rotarod test from the mice treated with siTRIM21@RVG-PLGA NPs or siNC@RVG-PLGA NPs on days 1, 3, 5, and 7 post-tMCAO ( n = 8 to 14, 2-way ANOVA followed by Tukey’s post hoc test). All data are expressed as means ± SD. * P < 0.05 and ** P < 0.01 versus indicated groups; # P < 0.05 and ### P < 0.001 versus tMCAO + siTRIM21@RVG-PLGA NPs group.

Article Snippet: WT mice and Trim21 KO mice (NM-KO-190403) were obtained from Shanghai Model Organisms Center.

Techniques: Transmission Assay, Electron Microscopy, Fluorescence, In Vivo, Injection, Ex Vivo, Staining, Derivative Assay