trim21 ko mice  (Shanghai Model Organisms Center)

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