Journal: Advanced Science

Article Title: Macrophage TRIM21 Inhibition Ameliorates Murine Acute Pancreatitis via PHB2‐Mediated Mitochondrial Stabilization

doi: 10.1002/advs.202517877

Figure Lengend Snippet: TRIM21 expression was elevated in acute pancreatitis (AP). (A) Cross‐dataset analysis of TRIM family gene expression using three independent Gene Expression Omnibus (GEO) datasets. (B) Immunohistochemical (IHC) staining for TRIM21 in the pancreas from L‐arginine‐treated and saline‐treated mice (scale bar: 100 µm; n = 6). (C, D) Co‐immunofluorescence of TRIM21 (red) with F4/80 (green; macrophage marker) or MPO (green; neutrophil marker). Cell nuclei were stained with DAPI (scale bar: 25 µm; n = 5). (E) Representative Western blotting images (upper panel) and quantification (lower panel) of the expression levels of TRIM21 protein in circulating monocytes from L‐arginine‐treated and saline‐treated mice ( n = 6). (F) The plasma levels of TRIM21 in healthy controls ( n = 29) and those with mild AP ( n = 41), moderately severe AP ( n = 37), and severe AP ( n = 23). (G) Representative Western blotting images (upper panel) and quantification (lower panel) of TRIM21 levels in circulating monocytes from patients with AP ( n = 12 per subtype) and healthy controls ( n = 12). (H) RT‐qPCR analysis of the mRNA levels of TRIM21 ( n = 5) and (I) Western blotting of the protein levels of TRIM21 ( n = 6) in bone marrow‐derived macrophages (BMDMs) treated with acinar cell supernatants (ACS). Data are expressed as mean ± SEM (B, E–I), and statistical analyses were conducted using Welch's t‐test (B, H), Student's t‐test (E, I), Kruskal‐Wallis test with Dunn's post hoc test (F), and ANOVA with Tukey HSD post hoc test (G).

Article Snippet: TRIM21 was activated using vilazodone (25 mg/kg; MedChemExpress) and downregulated using quisinostat (10 mg/kg; MedChemExpress).

Techniques: Expressing, Gene Expression, Immunohistochemical staining, Immunohistochemistry, Saline, Immunofluorescence, Marker, Staining, Western Blot, Clinical Proteomics, Quantitative RT-PCR, Derivative Assay

Journal: Advanced Science

Article Title: Macrophage TRIM21 Inhibition Ameliorates Murine Acute Pancreatitis via PHB2‐Mediated Mitochondrial Stabilization

doi: 10.1002/advs.202517877

Figure Lengend Snippet: Macrophage Trim21 deficiency attenuated L‐arginine‐induced AP. (A–G) Trim21 M‐KO and Trim21 M‐WT mice were treated with either saline or L‐arginine for 72 h ( Trim21 M‐WT + Saline, Trim21 M‐WT + L‐arginine, Trim21 M‐KO + Saline, Trim21 M‐KO + L‐arginine; n = 10 per group). (A) Serum amylase levels. (B) Representative gross morphology of the pancreas. (C) Pancreas‐to‐body weight ratio. (D) Hematoxylin and eosin (H&E) staining (upper panel) and histopathological scoring (lower panel) of the pancreatic tissue. (E) Representative images (upper panel) and quantification (lower panel) of IHC staining for interleukin‐1 beta (IL‐1β) / tumor necrosis factor‐alpha (TNF‐α) in the pancreatic tissue. (F, G) IL‐1β and TNF‐α levels in serum samples and bronchoalveolar lavage fluid (BALF). Data are expressed as mean ± SEM (A, C–G), and statistical analyses were conducted using ANOVA with Tukey HSD post hoc test (A, TNF‐α in E), Welch's ANOVA with Games‐Howell post hoc test (C, IL‐1β in E, F, G), and Kruskal‐Wallis test with Dunn's post hoc test (D).

Article Snippet: TRIM21 was activated using vilazodone (25 mg/kg; MedChemExpress) and downregulated using quisinostat (10 mg/kg; MedChemExpress).

Techniques: Saline, Staining, Immunohistochemistry

Journal: Advanced Science

Article Title: Macrophage TRIM21 Inhibition Ameliorates Murine Acute Pancreatitis via PHB2‐Mediated Mitochondrial Stabilization

doi: 10.1002/advs.202517877

Figure Lengend Snippet: Vilazodone‐mediated activation of TRIM21 exacerbated L‐arginine‐induced AP. (A‐G) Trim21 M‐WT mice pretreated with vehicle or vilazodone intraperitoneally received saline or L‐arginine for 72 h (Vehicle + Saline, Vehicle + L‐arginine, Vilazodone + Saline, Vilazodone + L‐arginine; n = 10 per group). (A) Serum amylase levels. (B) Representative gross morphology of the pancreas. (C) Pancreas‐to‐body weight ratio. (D) H&E staining (upper panel) and histopathological scores (lower panel) of the pancreatic tissue. (E) Representative images (upper panel) and quantification (lower panel) of IHC staining for IL‐1β and TNF‐α in pancreatic tissue. (F, G) IL‐1β and TNF‐α levels in serum samples and BALF. Data are expressed as mean ± SEM (A, C–G), and statistical analyses were conducted using ANOVA with Tukey HSD post hoc test (A, C), Kruskal‐Wallis test with Dunn's post hoc test (D), and Welch's ANOVA with Games‐Howell post hoc test (E–G).

Article Snippet: TRIM21 was activated using vilazodone (25 mg/kg; MedChemExpress) and downregulated using quisinostat (10 mg/kg; MedChemExpress).

Techniques: Activation Assay, Saline, Staining, Immunohistochemistry

Journal: Advanced Science

Article Title: Macrophage TRIM21 Inhibition Ameliorates Murine Acute Pancreatitis via PHB2‐Mediated Mitochondrial Stabilization

doi: 10.1002/advs.202517877

Figure Lengend Snippet: Macrophage TRIM21 regulates PHB2‐mediated cGAS/STING signaling pathways. (A) Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of differentially expressed proteins (DEPs) in pancreatic tissues from L‐arginine‐induced Trim21 M‐WT and Trim21 M‐KO mice ( n = 3). The bubble diagram represents the number of genes under a specific term. The color of the dots represents the p ‐value. (B, C) Representative Western blotting images and quantification of the expression levels of mitophagy‐associated proteins (PINK1, Parkin, p62, and LC3B) in BMDMs from Trim21 M‐WT and Trim21 M‐KO mice, or Trim21 M‐WT mice transfected with an adenovirus carrying Trim21 (Ad‐ Trim21 ) and then stimulated with ACS for 12 h ( n = 5). (D) Co‐immunoprecipitation (co‐IP) with liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) analysis of pancreatic tissues from L‐arginine‐induced AP and saline‐induced control, with anti‐TRIM21 antibody or IgG antibody ( n = 3). (E) Intersection analysis of the DEPs associated with mitophagy and cytosolic DNA‐sensing pathways with the exclusive LC‐MS/MS signature proteins. (F, G) Western blotting of PHB2 in BMDMs from Trim21 M‐WT and Trim21 M‐KO mice, or Trim21 M‐WT mice transfected with Ad‐ Trim21 and then stimulated with ACS for 12 h ( n = 5). (H) Representative immunofluorescence images of mitophagic events in BMDMs from Trim21 M‐WT or Trim21 M‐KO mice after 12 h of treatment with ACS. Mitochondria (TOMM20, red) colocalizing with the autophagic marker LC3B (visualized using adenoviral mCherry‐GFP‐LC3B fusion protein) were identified as mitophagic events (indicated by the red arrows; scale bar: 10 µm; n = 5). (I) Representative immunofluorescence images of cytosolic mtDNA accumulation in BMDMs from Trim21 M‐WT or Trim21 M‐KO mice after 12 h of treatment with ACS. Areas where TFAM (mitochondrial transcription factor A, green) and double‐stranded DNA (dsDNA, red) signals overlapped, but did not co‐localize with mitochondria (TOMM20, yellow) or the nucleus (DAPI, blue), were identified as cytosolic mtDNA (indicated by the red arrows; scale bar: 10 µm; n = 5). (J) RT‐qPCR of cytosolic mtDNA in BMDMs from Trim21 M‐WT or Trim21 M‐KO mice after 12 h of treatment with ACS ( n = 5). (K) Representative Western blot images (left panel) and quantification (right panel) of mitophagy‐associated protein levels (PINK1, Parkin, p62, and LC3B) in BMDMs from Trim21 M‐WT or Trim21 M‐KO mice following transfection with PHB2 siRNA (si‐ Phb2 ) and subsequent 12‐h ACS treatment ( n = 5). (L) Representative immunofluorescence images of mitophagic events in BMDMs from Trim21 M‐WT or Trim21 M‐KO mice after transfection with Phb2 siRNA (si‐ Phb2 ) and 12 h of treatment with ACS. TOMM20 (red) colocalizing with the autophagic marker LC3B (visualized using adenoviral mCherry‐GFP‐LC3B fusion protein) was identified as a mitophagic event (indicated by the red arrows; scale bar: 10 µm; n = 5). (M) Representative immunofluorescence images (indicated by the red arrows; scale bar: 10 µm; n = 5) and (N) RT‐qPCR ( n = 5) of cytosolic mtDNA in BMDMs from Trim21 M‐WT or Trim21 M‐KO mice after transfection with si‐ Phb2 and 12 h of treatment with ACS. Data are expressed as mean ± SEM (B, C, F, G, J, K, N). Statistical analyses were conducted using ANOVA with Tukey HSD post hoc test (indicators other than p62 in B, C, F, G, K, N), Welch's ANOVA with Games‐Howell post hoc test (p62 in B), and Student's t‐test (J).

Article Snippet: TRIM21 was activated using vilazodone (25 mg/kg; MedChemExpress) and downregulated using quisinostat (10 mg/kg; MedChemExpress).

Techniques: Protein-Protein interactions, Western Blot, Expressing, Transfection, Immunoprecipitation, Co-Immunoprecipitation Assay, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Saline, Control, Immunofluorescence, Marker, Quantitative RT-PCR

Journal: Advanced Science

Article Title: Macrophage TRIM21 Inhibition Ameliorates Murine Acute Pancreatitis via PHB2‐Mediated Mitochondrial Stabilization

doi: 10.1002/advs.202517877

Figure Lengend Snippet: TRIM21 could bind to PHB2 via its coiled‐coil domain and promoted K11‐linked ubiquitination‐dependent proteasomal degradation. (A) Co‐IP with anti‐TRIM21 (left) or anti‐PHB2 (right) in BMDMs (n = 5). (B) Co‐localization of TRIM21 (red) with PHB2 (green) in BMDMs. Cell nuclei were stained with DAPI (blue) (scale bar: 10 µm; n = 5). (C) Domain diagrams of TRIM21 WT and deletion mutants. (D) Co‐IP analysis of the interactions of Flag‐PHB2 with His‐TRIM21 (WT or mutants) in HEK293T cells ( n = 5). (E) Representative Western blotting images (left panel) and quantification (right panel) of PHB2 levels in BMDMs transfected with gradient concentrations of Ad‐ Trim21 ( n = 5). (F) Representative Western blotting images (upper panel) and quantification (lower panel) of PHB2 levels in Ad‐ Trim21 ‐expressing BMDMs pretreated with chloroquine (CQ; 50 µ m ), 3‐methyladenine (3‐MA; 10 m m ), and Z‐Leu‐Leu‐Leu‐al (MG132; 10 µ m ) for 12 h ( n = 5). (G) Representative Western blotting images (upper panel) and quantification (lower panel) of PHB2 levels in BMDMs from Trim21 M‐WT or Trim21 M‐KO mice treated with cycloheximide (CHX; 100 µg/mL) or MG132 (10 µ m ) for 0 h or 12 h ( n = 5). (H) Co‐IP results for PHB2 ubiquitination levels. BMDMs from Trim21 M‐WT or Trim21 M‐KO mice were treated with MG132 (10 µ m ) for 12 h and with ACS for 12 h ( n = 5). (I) Co‐IP results for exogenous PHB2 ubiquitination levels. HEK293T cells were transfected with the control vector, His‐TRIM21(WT), Flag‐PHB2, and MYC‐ubiquitin (WT/K11/K48/K63) ( n = 5). Data are expressed as mean ± SEM (E–G). Statistical analyses were conducted using ANOVA with Tukey HSD post hoc test (E, F) and Kruskal‐Wallis test with Dunn's post hoc test (G).

Article Snippet: TRIM21 was activated using vilazodone (25 mg/kg; MedChemExpress) and downregulated using quisinostat (10 mg/kg; MedChemExpress).

Techniques: Ubiquitin Proteomics, Co-Immunoprecipitation Assay, Staining, Western Blot, Transfection, Expressing, Control, Plasmid Preparation

Journal: Advanced Science

Article Title: Macrophage TRIM21 Inhibition Ameliorates Murine Acute Pancreatitis via PHB2‐Mediated Mitochondrial Stabilization

doi: 10.1002/advs.202517877

Figure Lengend Snippet: Macrophage Phb2 deficiency reversed Trim21 deficiency‐mediated improvement in AP. (A–H) Trim21 M‐KO and Trim21 M‐WT mice received AAV9‐sh Phb2 or AAV9‐sh NC for 4 weeks, followed by 72 h of infusion of L‐arginine ( Trim21 M‐WT + AAV9‐sh NC + L‐arginine, Trim21 M‐WT + AAV9‐sh Phb2 + L‐arginine, Trim21 M‐KO + AAV9‐sh NC + L‐arginine, Trim21 M‐KO + AAV9‐sh Phb2 + L‐arginine, n = 10 per group). (A) Serum amylase levels. (B) Gross morphology of the pancreas. (C) Pancreas‐to‐body weight ratio. (D) H&E staining (upper panel) and histopathological scores (lower panel) of the pancreas. Representative images (E) and quantification (F) of IHC staining for IL‐1β and TNF‐α in the pancreatic tissue. (G, H) Serum and BALF levels of IL‐1β and TNF‐α. Data are expressed as mean ± SEM (A, C, D, F–H), and statistical analyses were conducted using Welch's ANOVA with Games‐Howell post hoc test (A, F), ANOVA with Tukey HSD post hoc test (C, G, H), and Kruskal‐Wallis test with Dunn's post hoc test (D).

Article Snippet: TRIM21 was activated using vilazodone (25 mg/kg; MedChemExpress) and downregulated using quisinostat (10 mg/kg; MedChemExpress).

Techniques: Staining, Immunohistochemistry

Journal: Advanced Science

Article Title: Macrophage TRIM21 Inhibition Ameliorates Murine Acute Pancreatitis via PHB2‐Mediated Mitochondrial Stabilization

doi: 10.1002/advs.202517877

Figure Lengend Snippet: TRIM21 suppression mitigated L‐arginine‐induced development of AP. (A–I) Trim21 M‐WT mice pretreated with vehicle or quisinostat intraperitoneally received L‐arginine for 72 h (AP, quisinostat + AP; n = 10 per group). (A) Serum amylase levels. (B) Gross morphology of the pancreas. (C) Pancreas‐to‐body weight ratio. H&E staining (D) and histopathological scores (E) of the pancreas. Representative images (F) and quantification (G) of IHC staining for IL‐1β and TNF‐α in pancreatic tissue. (H, I) IL‐1β and TNF‐α levels in serum samples and BALF. Data are expressed as mean ± SEM (A, C, E, G–I), and statistical analyses were conducted using Student's t‐test (A, C, IL‐1β in G, TNF‐α in I), Mann‐Whitney U test (E, H, IL‐1β in I), and Welch's t‐test (TNF‐α in G).

Article Snippet: TRIM21 was activated using vilazodone (25 mg/kg; MedChemExpress) and downregulated using quisinostat (10 mg/kg; MedChemExpress).

Techniques: Staining, Immunohistochemistry, MANN-WHITNEY

Journal: Advanced Science

Article Title: Macrophage TRIM21 Inhibition Ameliorates Murine Acute Pancreatitis via PHB2‐Mediated Mitochondrial Stabilization

doi: 10.1002/advs.202517877

Figure Lengend Snippet: Proposed working model of TRIM21. Macrophage‐specific TRIM21 upregulation exacerbates the development of AP. Mechanistically, macrophage TRIM21 facilitates PHB2 degradation through ubiquitin‐proteasome processing, leading to cytosolic mtDNA accumulation via impaired PHB2‐mediated mitophagy. This mtDNA homeostasis disruption triggers cGAS‐STING axis activation, thereby exacerbating inflammatory cascades during AP progression.

Article Snippet: TRIM21 was activated using vilazodone (25 mg/kg; MedChemExpress) and downregulated using quisinostat (10 mg/kg; MedChemExpress).

Techniques: Ubiquitin Proteomics, Disruption, Activation Assay

Journal: Journal of neuroinflammation

Article Title: Regulatory role of TRIM21 in the type-I interferon pathway in Japanese encephalitis virus-infected human microglial cells.

doi: 10.1186/1742-2094-11-24

Figure Lengend Snippet: Figure 1 TRIM21 attenuates the JEV mediated upregulation of the p-IRF3 level and IFN-β level in human microglial cells. (A) PCR amplification of TRIM21 and TRIM21 (ΔRING) primers was carried out and the product run on 1% agarose gel (upper panel). Expression of wild-type TRIM21 as well as the TRIM21 (ΔRING) domain was confirmed by Western blotting (lower panel). (B) CHME3 cells were transfected with 4 μg of TRIM21 plasmid or TRIM21 (ΔRING) for 48 h. Cell lysates were resolved on SDS-PAGE and probed with anti-TRIM21, anti-IRF-3 and anti-β- tubulin antibodies by Western blotting. Representative image is shown. (C) Cells transfected with TRIM21 or TRIM21 (ΔRING) were infected with JEV, and total RNA was isolated post 48 h of transfection. Real-time PCR for IFN-β1 was performed, and an average of three independent sets of experiments is plotted and shown. (D) Luciferase assay for IFN-β for cells transfected with TRIM21 or TRIM21 (ΔRING) and infected with JEV along with respective controls was performed. Luciferase activity normalized against β-gal activity was averaged and plotted (*p <0.05, **p < 0.01, ***p < 0.001 from control).

Article Snippet: SiRNA against TRIM21 was purchased from Origene (Rockville, MD, USA) along with Negative control scrambled RNA (TRIM21 Trilencer-27 Human siRNA; #SR304594).

Techniques: Amplification, Agarose Gel Electrophoresis, Expressing, Western Blot, Transfection, Plasmid Preparation, SDS Page, Infection, Isolation, Real-time Polymerase Chain Reaction, Luciferase, Activity Assay, Control

Journal: Journal of neuroinflammation

Article Title: Regulatory role of TRIM21 in the type-I interferon pathway in Japanese encephalitis virus-infected human microglial cells.

doi: 10.1186/1742-2094-11-24

Figure Lengend Snippet: Figure 4 JEV induces TRIM21 protein levels in human microglial cells in a time-dependent manner. (A) CHME3 cells were either un-infected or infected with JEV at MOI 5 for 6, 12, 24 and 36 h. Cell lysates were resolved on SDS-PAGE and probed with anti-TRIM21 antibody and anti-β- tubulin antibody (loading control) by Western blotting. A representation of three independent experiments is shown along with densitometry analysis with normalization of TRIM21 against β-tubulin, averaging and plotting. (B) CHME3 cells were infected with JEV for 24 h (MOI 5). Total RNA was isolated from harvested cells. cDNA prepared by reverse transcription of control and infected samples was used as a template for qPCR against primers for TRIM21 gene. Average fold change in the TRIM21 mRNA level from three independent experiments is plotted and shown (*p < 0.05, **p < 0.01, ***p < 0.001 from control, #p from 6 h, $p from 12 h).

Article Snippet: SiRNA against TRIM21 was purchased from Origene (Rockville, MD, USA) along with Negative control scrambled RNA (TRIM21 Trilencer-27 Human siRNA; #SR304594).

Techniques: Infection, SDS Page, Control, Western Blot, Isolation, Reverse Transcription

Journal: Journal of neuroinflammation

Article Title: Regulatory role of TRIM21 in the type-I interferon pathway in Japanese encephalitis virus-infected human microglial cells.

doi: 10.1186/1742-2094-11-24

Figure Lengend Snippet: Figure 5 TRIM21 knockdown facilitates JEV-mediated IRF3 activation and upregulation of the IFN-β level. (A) Cells were either transfected with negative control RNA (NC) or transfected with 10nM siRNA against TRIM21 for 48 h. Cell lysates were resolved on SDS-PAGE and probed with anti-TRIM21 antibody and anti-β-tubulin antibody by Western blotting. A representative image is shown. (B) Cells were either non-transfected (C), transfected with negative control RNA (NC) or with TRIM21 siRNA for 24 h followed by JEV infection for 24 h. Cell lysates were resolved on SDS-PAGE and probed with anti-p-IRF3 antibody, anti-IRF3 and anti-β-tubulin antibodies (loading control) by Western blotting. A representative of three independent experiments is shown. Densitometry analyses of Western blot experiments were performed with normalizing p-IRF-3 and p-IRF-3 against β-tubulin. (C) Real-time PCR for IFN-β1 for siRNA-transfected and JEV-infected cells along with the respective controls was performed and averaged for three independent sets of experiments. (D) Luciferase assay for IFN-β for cells transfected with siRNA against TRIM21 and infected with JEV along with respective controls was performed. Luciferase activity normalized against β-gal activity was averaged and plotted (**p < 0.01, ***p < 0.001 from control).

Article Snippet: SiRNA against TRIM21 was purchased from Origene (Rockville, MD, USA) along with Negative control scrambled RNA (TRIM21 Trilencer-27 Human siRNA; #SR304594).

Techniques: Knockdown, Activation Assay, Transfection, Negative Control, SDS Page, Western Blot, Infection, Control, Real-time Polymerase Chain Reaction, Luciferase, Activity Assay

Journal: Journal of neuroinflammation

Article Title: Regulatory role of TRIM21 in the type-I interferon pathway in Japanese encephalitis virus-infected human microglial cells.

doi: 10.1186/1742-2094-11-24

Figure Lengend Snippet: Figure 6 Model showing a plausible role of TRIM21 as a negative regulator of IRF3 activation and IFN-β production following JEV infection in human microglial cells. JEV infection causes activation of the RIG-1 receptor, initiating a downstream signaling mechanism leading to the activation of IRF-3. Phosphorylated IRF-3 dimerizes and translocates into the nucleus, where it leads to the transcription and production of IFN-β. JEV infection also induces the TRIM21 protein, which negatively regulates IRF-3 phosphorylation, leading to reduced IFN-β production. The upregulation of TRIM21 is proposed to be a feedback mechanism to inhibit the innate immune response in JEV infection.

Article Snippet: SiRNA against TRIM21 was purchased from Origene (Rockville, MD, USA) along with Negative control scrambled RNA (TRIM21 Trilencer-27 Human siRNA; #SR304594).

Techniques: Activation Assay, Infection, Phospho-proteomics

Journal: Scientific Reports

Article Title: Leukocyte immunoglobulin-like receptor B4 regulates key signalling molecules involved in FcγRI-mediated clathrin-dependent endocytosis and phagocytosis

doi: 10.1038/srep35085

Figure Lengend Snippet: Mascot search results of mass spectrometric peptides sequencing of tyrosine phosphorylated proteins following FcγRI cross linking of THP-1 cells (n = 3).

Article Snippet: The following antibodies were used for Western blotting: biotinylated mouse α-pTyr-100 mAb (Cell Signaling, Danvers, MA, USA), mouse anti-human clathrin (Thermo Fisher Scientific, Waltham, MA, USA), mouse anti-HSP70 (Stressgen/Enzo Life Sciences, Farmingdale, NY, USA), rabbit anti-HGS (Thermo Fisher Scientific), rabbit anti-Cbl (Sigma-Aldrich), rabbit anti-FcγRs (Upstate Biotechnology Inc, Lake placid, NY, USA), rabbit anti-Syk (Cell Signaling), rabbit anti-pSyk (Tyr 525/526) (Cell Signaling), mouse anti-β-actin (Sigma-Aldrich), and mouse anti-human TRIM21 mAb (R&D System) in-house labelled with biotin using lightning-link TM biotin conjugation kit (Innova Biosciences, Babraham, Cambridge, UK), primary antibodies, and HRP-conjugated goat anti-mouse or goat anti-rabbit secondary antibodies, and HRP-conjugated streptavidin (all from Bio-Rad, Gladesville, NSW, Australia).

Techniques: Sequencing, Control, Ubiquitin Proteomics, Binding Assay, Variant Assay, Activation Assay

Journal: Scientific Reports

Article Title: Leukocyte immunoglobulin-like receptor B4 regulates key signalling molecules involved in FcγRI-mediated clathrin-dependent endocytosis and phagocytosis

doi: 10.1038/srep35085

Figure Lengend Snippet: ( A ) Representative immunoprecipitation of THP-1 cell lysates using anti-pTyr mAb (4G10) followed by Western blotting using selected antibodies showed abundant Tyr phosphorylation of FcγR s, clatherin, HSP70, Cbl, HGS and TRIM21 in cells co-ligated with anti-FcγRI+IgG 1 control mAb validating LC-MS/MS data. Importantly, LILRB4 co-ligation with FcγRI markedly reduced Tyr phosphorylation of all proteins except for HSP70 (n = 3). ( B ) Summary of densitometry of bands from 3 independent experiments showed significant reduction of FcγRs, clatherin, Cbl, HGS and TRIM21 phosphorylation, but not HSP70, in THP-1 cells co-ligated with anti-FcγRI and anti-LILRB4 mAbs, compared to cells co-ligated with anti-FcγRI and negative control mAb (n = 3, **p < 0.01; ***p < 0.001). ( C ) Representative Western blotting of total cell lysates showed that co-ligation of FcγRI with LILRB4 did not alter the total amounts of any of the above proteins when compared to co-ligation of FcγRI+IgG 1 control, ligation of LILRB4 alone or treatment with IgG 1 control alone; the lower panel is the same membrane stripped and re-probed with anti-β actin Ab, confirming comparable protein loading. ( D ) Summary of densitometry analysis of 3 independent experiments showed no significant differences in total FcγRs, clatherin, HSP70, Cbl, HGS and TRIM21 in THP-1 cells within the 4 different treatment groups (n = 3). Full image of the Western blots is shown in . ( E ) Western blotting of cell lysates from FcγRI cross-linked cells showing increased Tyr-phosphorylated Syk that was markedly reduced upon co-ligation with LILRB4, confirming our earlier finding and validating current LC-MS/MS data (n = 1).

Article Snippet: The following antibodies were used for Western blotting: biotinylated mouse α-pTyr-100 mAb (Cell Signaling, Danvers, MA, USA), mouse anti-human clathrin (Thermo Fisher Scientific, Waltham, MA, USA), mouse anti-HSP70 (Stressgen/Enzo Life Sciences, Farmingdale, NY, USA), rabbit anti-HGS (Thermo Fisher Scientific), rabbit anti-Cbl (Sigma-Aldrich), rabbit anti-FcγRs (Upstate Biotechnology Inc, Lake placid, NY, USA), rabbit anti-Syk (Cell Signaling), rabbit anti-pSyk (Tyr 525/526) (Cell Signaling), mouse anti-β-actin (Sigma-Aldrich), and mouse anti-human TRIM21 mAb (R&D System) in-house labelled with biotin using lightning-link TM biotin conjugation kit (Innova Biosciences, Babraham, Cambridge, UK), primary antibodies, and HRP-conjugated goat anti-mouse or goat anti-rabbit secondary antibodies, and HRP-conjugated streptavidin (all from Bio-Rad, Gladesville, NSW, Australia).

Techniques: Immunoprecipitation, Western Blot, Phospho-proteomics, Control, Liquid Chromatography with Mass Spectroscopy, Ligation, Negative Control, Membrane

Journal: Scientific Reports

Article Title: Leukocyte immunoglobulin-like receptor B4 regulates key signalling molecules involved in FcγRI-mediated clathrin-dependent endocytosis and phagocytosis

doi: 10.1038/srep35085

Figure Lengend Snippet: Cross-linking of FcγRI by immune-complexes causes Tyr phosphorylation of the ITAMs of its common γ chain and binding of pSyk transduces activating signals. This simultaneously initiates phosphorylation of clatherin that causes lateral diffusion of receptor-ligand complexes to clathrin-coated pits, membrane invagination and generation of clathrin-coated vesicles, and/or initiates phosphorylation of Cbl that may directly ubiquitinate the receptor. Phosphorylated Cbl triggers phosphorylation of HSP70 that facilitates un-coating of the vesicles, a precondition for vesicles to fuse with early endosomes and release ligands. The released receptors are transported to either the late endosome and/or lysosome for proteosomal and/or lysosomal degradation or are recycled to the cell surface. The immune complexes in the endosome are either directly degraded by Cbl, or delivered to the lysosome by phosphorylated HGS-STAM 1/2 complex for final degradation. During transfer, immune complexes that escape the endosome are recognised by phosphorylated TRIM21 for proteasomal degradation. Co-ligation of FcγRI with LILRB4 may recruit phosphatases such as SHP-1 to its ITIMs that subsequently dephosphorylate (deactivate) the key molecules including clathrin (1), FcγRI and Syk (2), Cbl (3), HGS and STAM 1/2 (4) and TRIM21(5). These effects may reduce cellular activation and/or suppress receptor/ligand endocytosis. *New Tyr phosphorylated and dephosphorylated proteins identified in this study.

Article Snippet: The following antibodies were used for Western blotting: biotinylated mouse α-pTyr-100 mAb (Cell Signaling, Danvers, MA, USA), mouse anti-human clathrin (Thermo Fisher Scientific, Waltham, MA, USA), mouse anti-HSP70 (Stressgen/Enzo Life Sciences, Farmingdale, NY, USA), rabbit anti-HGS (Thermo Fisher Scientific), rabbit anti-Cbl (Sigma-Aldrich), rabbit anti-FcγRs (Upstate Biotechnology Inc, Lake placid, NY, USA), rabbit anti-Syk (Cell Signaling), rabbit anti-pSyk (Tyr 525/526) (Cell Signaling), mouse anti-β-actin (Sigma-Aldrich), and mouse anti-human TRIM21 mAb (R&D System) in-house labelled with biotin using lightning-link TM biotin conjugation kit (Innova Biosciences, Babraham, Cambridge, UK), primary antibodies, and HRP-conjugated goat anti-mouse or goat anti-rabbit secondary antibodies, and HRP-conjugated streptavidin (all from Bio-Rad, Gladesville, NSW, Australia).

Techniques: Phospho-proteomics, Binding Assay, Diffusion-based Assay, Membrane, Ligation, Activation Assay

Journal: Science advances

Article Title: CLEC-1 is a death sensor that limits antigen cross-presentation by dendritic cells and represents a target for cancer immunotherapy.

doi: 10.1126/sciadv.abo7621

Figure Lengend Snippet: Fig. 2. Identification of TRIM21 as a ligand of CLEC-1. (A) Representative affinity chromatography elution products (SDS–polyacrylamide gel electrophoresis) after co-IP of UV-treated Raji or Hpb-all whole-cell extract (WCE) with protein G beads coupled to human Fc CLEC-1 or Fc alone (control) (three independent experiments). Arrowheads at 50 kDa indicate a band only present under the Fc CLEC-1 condition. MW, molecular weight. (B) LC-MS analysis (of the 50-kDa gel band) depicting the top hits of Fc CLEC-1 over the Fc alone control condition (n = 4, mean). (C) Representative Western blot of human TRIM21 after co-IP of UV-treated Raji WCE (or without cells as control) with protein G beads coupled to human Fc CLEC-1 or irrelevant Fc CLEC7A (three independent experiments). Arrowheads correspond to TRIM21 detected in the co-IP in the retained and unretained fractions. (D) Representative Western blot of human CLEC-1 in elution product after co-IP of UV-treated Raji WCE and His–CLEC-1 with protein G beads coupled to antihuman TRIM21 mAb and revealed with either anti-His or anti–CLEC-1 mAb (three independent experiments). (E) Representative Western blot of human TRIM21 after co-IP of UV-treated Raji transduced with lentiviral TRIM21 (sh+) or control scrambled shRNA with protein G beads coupled to human Fc CLEC-1 (three independent experiments). (F) Flow cytometry analysis of human Fc CLEC-1 binding on UV-treated Raji transduced with lentiviral TRIM21 or control scrambled shRNA (Ctrl) (data expressed as fold change of binding of Fc CLEC-1 compared to binding of Fc alone) (n = 5, paired t test, *P < 0.05). (G) Sensograms generated by Biacore-based affinity measurement representing interaction of chip-immobilized recombinant human TRIM21 with five increasing concentrations of human Fc CLEC-1 (Fc alone as control) or His–CLEC-1 [irrelevant (irr) protein as control]. (H) Representative ELISA [density of optical (DO)] of recombinant human TRIM21 on coated human Fc CLEC-1 or Fc alone (n = 4, means ± SEM, unpaired t test, **P < 0.01 and ***P < 0.001).

Article Snippet: Reverse co-IP was performed as co-IP, except that beads were coated with anti-TRIM21 antibody (R&D Systems) or control isotype (BD Pharm) and coincubated with His CLEC-1 protein (Bio-Techne).

Techniques: Affinity Chromatography, Polyacrylamide Gel Electrophoresis, Co-Immunoprecipitation Assay, Control, Molecular Weight, Liquid Chromatography with Mass Spectroscopy, Western Blot, Transduction, shRNA, Flow Cytometry, Binding Assay, Generated, Recombinant, Enzyme-linked Immunosorbent Assay

Journal: Science advances

Article Title: CLEC-1 is a death sensor that limits antigen cross-presentation by dendritic cells and represents a target for cancer immunotherapy.

doi: 10.1126/sciadv.abo7621

Figure Lengend Snippet: Fig. 6. Treatment with antihuman CLEC-1 antagonist mAb enhances antitumor immunity in CLEC-1 humanized mice. (A) CLEC1A or Clec1a mRNA expression in different subsets of myeloid cells from human or mouse lung tumors from Open-source data of scRNA-seq. AU, arbitrary units. (B) Two-dimensional Uniform Manifold Approximation and Projection and violin plot representations of CLEC1A expression (red scale projection) among clusters of myeloid cells and DCs from scRNA-seq data- set of pan-cancer (breast, colorectal, lung, and ovarian) TME. (C) Box plots representation of log2 [transcript count per million (TMP)+1] values for TRIM21 expression in dif- ferent cancer types from TCGA analyzed by GEPIA tool. BRCA, breast-invasive carcinoma; CESC, cervical squamous cell carcinoma and endocervical adenocarcinoma; CHOL, cholangiocarcinoma; COAD, colon adenocarcinoma; ESCA, esophageal carcinoma; GBM, glioblastoma multiforme; HNSC, head and neck squamous cell carcinoma; KIRC, kidney renal clear cell carcinoma; LAML, acute myeloid leukemia; LGG, brain lower-grade glioma; LIHC, liver hepatocellular carcinoma; PAAD, pancreatic adenocarcinoma; READ, rectum adenocarcinoma; SKCM, skin cutaneous melanoma; STAD, stomach adenocarcinoma; TGCT, testicular germ cell tumors. Unpaired t test, *P < 0.05. (D and E) Tumor growth or survival curve of CLEC-1 humanized mice with subcutaneous MC38 adenocarcinoma in combination with CPA (150 mg/kg) (D) or with Hepa 1.6 LIHC (E) and treated with irrelevant isotype control or antihuman CLEC-1 antagonist mAbs #1 or #2 (100 g per mouse) twice a week from days 4 to 21 (n = 14 to 17 of two independent experiments, unpaired t test, *P < 0.05 for tumor growth, and log-rank test, *P < 0.05 and **P < 0.01 for survival). (F) Percentage of PMNs/MDSCs (CD11b+Ly6G+MHC-II−), macrophages (CD11b+F4/80+Ly6C+), DCs (CD11c+MHC-II+), CD69+or Tmem (CD44+) CD8+, or CD4+ (CD3+CD4/8+) T cells evaluated by flow cytometry at day 13 after LIHC in the tumor- burdened livers of CLEC-1 humanized mice treated with isotype control or antihuman CLEC-1 antagonist mAbs #4 (100 g per mouse) (data are expressed in the percentage of CD45+ or in MFI of MHC-II expression) (n = 8 to 13, means ± SEM of three independent experiments, unpaired t test *P < 0.05, **P < 0.01, and ***P < 0.001).

Article Snippet: Reverse co-IP was performed as co-IP, except that beads were coated with anti-TRIM21 antibody (R&D Systems) or control isotype (BD Pharm) and coincubated with His CLEC-1 protein (Bio-Techne).

Techniques: Expressing, Control, Flow Cytometry

Journal: Life science alliance

Article Title: FAT10 inhibits TRIM21 to down-regulate antiviral type-I interferon secretion.

doi: 10.26508/lsa.202402786

Figure Lengend Snippet: Figure 1. TRIM21 is covalently modified with FAT10. (A) HEK293T cells were transiently transfected with expression constructs for HA-FAT10 and Myc-DDK-TRIM21. After 24 h, cells were harvested and lysed. Cleared lysate was subjected to immunoprecipitation (IP) using FLAG M2 affinity gel, which specifically recognizes the DDK (FLAG) tag. Proteins were visualized by Western blot analysis under reducing conditions (4% 2-ME) using antibodies reactive to HA or FLAG (DDK). GAPDH was used as a loading control. (B) HEK293T cells were transiently co-transfected

Article Snippet: Plasmids used for the transient transfection of HEK293 cells were pCMV6-Myc-DDK-TRIM21 (RC202088; Origene), pcDNA3.1-HA-FAT10 (Hipp et al, 2004), pcDNA3.1-HA-FAT10AV (Aichem et al, 2010), pcDNA3.1-HA-UBA6 (Aichem et al, 2010), pcDNA-3.1-His/-A-USE1, and its active site cysteine mutant pcDNA-3.1-His/-A-USE1-C188A (Aichem et al, 2010), lentiviral envelop plasmid pMD2.G (#12259; Addgene), and lentiviral packaging plasmid psPAX2 (#12260; Addgene). pSMPP-mCherry-hTRIM21 was a gift from Leo James (plasmid #104972 [Clift et al, 2017]; Addgene), and pSpCAS9 wt (BB)2A-GFP targeting human TRIM21 was a gift from Gaudenz Danuser (plasmid # 138295 [Park et al, 2020]; Addgene). pCMV6-Myc-DDKTRIM21 (RC202088; Origene) was used as template to construct the truncation mutations of TRIM21 by site-directed mutagenesis.

Techniques: Transfection, Expressing, Construct, Immunoprecipitation, FLAG-tag, Western Blot, Control

Journal: Life science alliance

Article Title: FAT10 inhibits TRIM21 to down-regulate antiviral type-I interferon secretion.

doi: 10.26508/lsa.202402786

Figure Lengend Snippet: Figure 2. FAT10ylation of TRIM21 is catalyzed by the E1 UBA6 and the E2 USE1. (A) HEK293 WT, or where indicated UBA6 KO and USE1 KO cells, were transiently transfected with HA–FAT10 or HA–FAT10AV and Myc–DDK–TRIM21 expression plasmids. After 24 h, cells were collected and lysed. Cleared cell lysate was subjected to SDS–PAGE and subsequent Western blotting under reducing conditions (4% 2-ME) using antibodies reactive to HA or FLAG (DDK). GAPDH was used as loading control. (B) HEK293 WT, or UBA6 KO and USE1 KO cells were transiently transfected with expression

Article Snippet: Plasmids used for the transient transfection of HEK293 cells were pCMV6-Myc-DDK-TRIM21 (RC202088; Origene), pcDNA3.1-HA-FAT10 (Hipp et al, 2004), pcDNA3.1-HA-FAT10AV (Aichem et al, 2010), pcDNA3.1-HA-UBA6 (Aichem et al, 2010), pcDNA-3.1-His/-A-USE1, and its active site cysteine mutant pcDNA-3.1-His/-A-USE1-C188A (Aichem et al, 2010), lentiviral envelop plasmid pMD2.G (#12259; Addgene), and lentiviral packaging plasmid psPAX2 (#12260; Addgene). pSMPP-mCherry-hTRIM21 was a gift from Leo James (plasmid #104972 [Clift et al, 2017]; Addgene), and pSpCAS9 wt (BB)2A-GFP targeting human TRIM21 was a gift from Gaudenz Danuser (plasmid # 138295 [Park et al, 2020]; Addgene). pCMV6-Myc-DDKTRIM21 (RC202088; Origene) was used as template to construct the truncation mutations of TRIM21 by site-directed mutagenesis.

Techniques: Transfection, Expressing, SDS Page, Western Blot, Control

Journal: Life science alliance

Article Title: FAT10 inhibits TRIM21 to down-regulate antiviral type-I interferon secretion.

doi: 10.26508/lsa.202402786

Figure Lengend Snippet: Figure 3. FAT10 targets TRIM21 for degradation by the 26S proteasome. (A) HEK293T cells were transiently transfected with expression constructs for HA–FAT10 and Myc–DDK–TRIM21. After 24 h, cells were treated with cycloheximide and/or MG132 for the indicated time points. Cleared cell lysates were subjected to immunoprecipitation using FLAG M2 affinity gel, which is specific for the DDK- tag. SDS–PAGE and Western blot analysis was performed under reducing conditions (4% 2-ME) using antibodies reactive to HA or FLAG (DDK). GAPDH was used as loading control. Shown is one representative experiment out of three independent experiments with similar outcomes. (B) Densitometric quantification of the FAT10-TRIM21 conjugate fluorescent signal, normalized to the respective GAPDH fluorescent signal. The value of untreated sample was set to 100%. Shown is the mean three independent experiments with similar outcomes. Source data are available for this figure.

Article Snippet: Plasmids used for the transient transfection of HEK293 cells were pCMV6-Myc-DDK-TRIM21 (RC202088; Origene), pcDNA3.1-HA-FAT10 (Hipp et al, 2004), pcDNA3.1-HA-FAT10AV (Aichem et al, 2010), pcDNA3.1-HA-UBA6 (Aichem et al, 2010), pcDNA-3.1-His/-A-USE1, and its active site cysteine mutant pcDNA-3.1-His/-A-USE1-C188A (Aichem et al, 2010), lentiviral envelop plasmid pMD2.G (#12259; Addgene), and lentiviral packaging plasmid psPAX2 (#12260; Addgene). pSMPP-mCherry-hTRIM21 was a gift from Leo James (plasmid #104972 [Clift et al, 2017]; Addgene), and pSpCAS9 wt (BB)2A-GFP targeting human TRIM21 was a gift from Gaudenz Danuser (plasmid # 138295 [Park et al, 2020]; Addgene). pCMV6-Myc-DDKTRIM21 (RC202088; Origene) was used as template to construct the truncation mutations of TRIM21 by site-directed mutagenesis.

Techniques: Transfection, Expressing, Construct, Immunoprecipitation, SDS Page, Western Blot, Control

Journal: Life science alliance

Article Title: FAT10 inhibits TRIM21 to down-regulate antiviral type-I interferon secretion.

doi: 10.26508/lsa.202402786

Figure Lengend Snippet: Figure 4. FAT10 modulates the stability of TRIM21 upon influenza A virus (IAV) infection. (A) A549 FLAG-FAT10 cells were infected with IAV (MOI: 1) as indicated and uninfected A549 cells were used as control. After 24 h, cells were harvested and lysed. Precleared cell lysates were subjected to Ni–IDA pull-down assay and incubated overnight at 4°C. Beads were washed four times with lysis buffer. SDS–PAGE under reducing conditions (4% 2-ME) followed by Western blotting was performed using the indicated antibodies. GAPDH was used as the loading control. Asterisk marks

Article Snippet: Plasmids used for the transient transfection of HEK293 cells were pCMV6-Myc-DDK-TRIM21 (RC202088; Origene), pcDNA3.1-HA-FAT10 (Hipp et al, 2004), pcDNA3.1-HA-FAT10AV (Aichem et al, 2010), pcDNA3.1-HA-UBA6 (Aichem et al, 2010), pcDNA-3.1-His/-A-USE1, and its active site cysteine mutant pcDNA-3.1-His/-A-USE1-C188A (Aichem et al, 2010), lentiviral envelop plasmid pMD2.G (#12259; Addgene), and lentiviral packaging plasmid psPAX2 (#12260; Addgene). pSMPP-mCherry-hTRIM21 was a gift from Leo James (plasmid #104972 [Clift et al, 2017]; Addgene), and pSpCAS9 wt (BB)2A-GFP targeting human TRIM21 was a gift from Gaudenz Danuser (plasmid # 138295 [Park et al, 2020]; Addgene). pCMV6-Myc-DDKTRIM21 (RC202088; Origene) was used as template to construct the truncation mutations of TRIM21 by site-directed mutagenesis.

Techniques: Virus, Infection, Control, Pull Down Assay, Incubation, Lysis, SDS Page, Western Blot

Journal: Life science alliance

Article Title: FAT10 inhibits TRIM21 to down-regulate antiviral type-I interferon secretion.

doi: 10.26508/lsa.202402786

Figure Lengend Snippet: Figure 5. FAT10-mediated inhibition of TRIM21 down-regulates IFNβ secretion upon influenza A virus (IAV) infection. (A) A549 WT, FLAG–FAT10, TRIM21 KO, TRIM21 KO/FLAG–FAT10 cells were infected with IAV (MOI: 1). After 24 h, supernatants and cell pellets were collected. Cell pellets were lysed and cleared cell lysates were subject to SDS–PAGE under reducing conditions (4% 2-ME) followed by Western blot analysis with the indicated antibodies. GAPDH was used as the loading control. (B) IFNβ ELISA was performed with the cell culture supernatants from (A). (C) A549 WT, FLAG–FAT10, mCherry-TRIM21, mCherry-

Article Snippet: Plasmids used for the transient transfection of HEK293 cells were pCMV6-Myc-DDK-TRIM21 (RC202088; Origene), pcDNA3.1-HA-FAT10 (Hipp et al, 2004), pcDNA3.1-HA-FAT10AV (Aichem et al, 2010), pcDNA3.1-HA-UBA6 (Aichem et al, 2010), pcDNA-3.1-His/-A-USE1, and its active site cysteine mutant pcDNA-3.1-His/-A-USE1-C188A (Aichem et al, 2010), lentiviral envelop plasmid pMD2.G (#12259; Addgene), and lentiviral packaging plasmid psPAX2 (#12260; Addgene). pSMPP-mCherry-hTRIM21 was a gift from Leo James (plasmid #104972 [Clift et al, 2017]; Addgene), and pSpCAS9 wt (BB)2A-GFP targeting human TRIM21 was a gift from Gaudenz Danuser (plasmid # 138295 [Park et al, 2020]; Addgene). pCMV6-Myc-DDKTRIM21 (RC202088; Origene) was used as template to construct the truncation mutations of TRIM21 by site-directed mutagenesis.

Techniques: Inhibition, Virus, Infection, SDS Page, Western Blot, Control, Enzyme-linked Immunosorbent Assay, Cell Culture

Journal: Life science alliance

Article Title: FAT10 inhibits TRIM21 to down-regulate antiviral type-I interferon secretion.

doi: 10.26508/lsa.202402786

Figure Lengend Snippet: Figure 6. Cartoon summarizing FAT10 mediated inhibition of type-I IFN signaling. TNF/IFNγ induces the expression of FAT10 (blue arrow). FAT10-mediated down-regulation of type-I IFN happens in two ways: (I) influenza A virus infection upregulates TRIM21 expression (blue arrow). TRIM21 positively regulates the antiviral type-I IFN production through a positive feedback loop (blue arrow). FAT10 inhibits TRIM21 by directly binding to its PRYSPRY domain, causing either degradation of TRIM21 by the 26S proteasome, and/or inhibiting TRIM21 auto- ubiquitination, thus down-regulating the production of type-I IFN. (II) FAT10 gets phosphorylated upon influenza A virus infection. Phosphorylated FAT10 stabilizes and activates OTUB1, which inhibits type-I IFN production (Saxena et al, 2024).

Article Snippet: Plasmids used for the transient transfection of HEK293 cells were pCMV6-Myc-DDK-TRIM21 (RC202088; Origene), pcDNA3.1-HA-FAT10 (Hipp et al, 2004), pcDNA3.1-HA-FAT10AV (Aichem et al, 2010), pcDNA3.1-HA-UBA6 (Aichem et al, 2010), pcDNA-3.1-His/-A-USE1, and its active site cysteine mutant pcDNA-3.1-His/-A-USE1-C188A (Aichem et al, 2010), lentiviral envelop plasmid pMD2.G (#12259; Addgene), and lentiviral packaging plasmid psPAX2 (#12260; Addgene). pSMPP-mCherry-hTRIM21 was a gift from Leo James (plasmid #104972 [Clift et al, 2017]; Addgene), and pSpCAS9 wt (BB)2A-GFP targeting human TRIM21 was a gift from Gaudenz Danuser (plasmid # 138295 [Park et al, 2020]; Addgene). pCMV6-Myc-DDKTRIM21 (RC202088; Origene) was used as template to construct the truncation mutations of TRIM21 by site-directed mutagenesis.

Techniques: Inhibition, Expressing, Virus, Infection, Binding Assay, Ubiquitin Proteomics

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: SUCLG2 Regulates Mitochondrial Dysfunction through Succinylation in Lung Adenocarcinoma.

doi: 10.1002/advs.202303535

Figure Lengend Snippet: Figure 5. TRIM21 induces SUCLG2 degradation through the K63-linkage ubiquitin lysosomal pathway. A) BEAS-2B, A549, HCC827, and H1299 cells were treated with 20 μg mL−1 CHX and collected at the indicated time. The degradation rate of SUCLG2 was tested by western blotting (left-hand panels). Relative SUCLG2 expression was analyzed using ImageJ (right-hand panel). Data represent the average of three independent experiments (mean ± SD). ***p < 0.001. B) CHX (20 μg mL−1) was added to A549 cells for 24 h, and CQ (20 μm) or MG132 (20 μm) was added at the same time. The expression of SUCLG2 was detected using western blotting. C) pcDNA3.1-His-SUCLG2 was overexpressed in A549 cells treated with MG132 (20 μm) or CQ (20 μm) for 24 h. Co-IP and western blotting were used to detect the ubiquitination level of SUCLG2. D) The indicated plasmids were transfected into A549 cells

Article Snippet: SUCLG2 siRNA (SR305801), SIRT5 siRNA (SR323578), and TRIM21 siRNA (SR321894) were ordered from Origene.

Techniques: Ubiquitin Proteomics, Western Blot, Expressing, Co-Immunoprecipitation Assay, Transfection

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: SUCLG2 Regulates Mitochondrial Dysfunction through Succinylation in Lung Adenocarcinoma.

doi: 10.1002/advs.202303535

Figure Lengend Snippet: Figure 6. The K93-succinylation of SUCLG2 affects the TRIM21-mediated degradation of SUCLG2. A) pcDNA-3.1-His-SUCLG2 was transfected into A549 cells, and co-IP and western blotting were used to detect the succinylation level of SUCLG2. B) The succinylation sites of SUCLG2 predicted by PhosphoSitePlus. C) A549 cells overexpressing the SUCLG2-WT or SUCLG2 lysine residue-mutated plasmids were prepared, and co-IP and western blotting were used to detect the succinylation of protein. D) A549 cells were transfected with the pcDNA-His-SUCLG2 or pcDNA-His-SUCLG2K93R

Article Snippet: SUCLG2 siRNA (SR305801), SIRT5 siRNA (SR323578), and TRIM21 siRNA (SR321894) were ordered from Origene.

Techniques: Transfection, Co-Immunoprecipitation Assay, Western Blot, Residue

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: SUCLG2 Regulates Mitochondrial Dysfunction through Succinylation in Lung Adenocarcinoma.

doi: 10.1002/advs.202303535

Figure Lengend Snippet: Figure 8. Inhibition of succinylation at the SUCLG2 K93 site leads to the mitochondrial dysfunction and reduced tumorigenesis of LUAD cells. A and B) LUAD cell lines A549 (A) and H1299 (B) were transfected with His-SUCLG2 and HA-TRIM21 plasmids. Twenty-four hours later, cells were seeded in 24-well plates. At the indicated times, cells were fixed with 4% paraformaldehyde and stained with 1% crystal violet. The dye was extracted with 10% acetic acid, and the relative proliferation was assessed based on the increase in absorbance at 595 nm (upper panels). Western blotting was used to determine the overexpression efficiency (bottom panels). The data represent the average of three independent experiments (mean ± SD). **p < 0.01, ***p < 0.001. C and D) LUAD cell lines A549 (C) and H1299 (D) were transfected with His-SUCLG2 and Flag-SIRT5 plasmids. Cell growth assays were

Article Snippet: SUCLG2 siRNA (SR305801), SIRT5 siRNA (SR323578), and TRIM21 siRNA (SR321894) were ordered from Origene.

Techniques: Inhibition, Transfection, Staining, Western Blot, Over Expression