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IFI35 ubiquitinates HNF4a in <t>TRIM21-dependent</t> 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

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1) Product Images from "IFI35 suppresses the transcription of hepatitis B virus cccDNA minichromosome via promoting HNF4α proteasomal degradation"

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

Journal: Journal of Biomedical Science

doi: 10.1186/s12929-026-01239-w

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

Figure Legend 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

Techniques Used: 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

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

Figure Legend 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

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

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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.

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

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.

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.

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

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.

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.

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

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.

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.

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

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.

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.

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

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.

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.

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

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.

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.

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

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

TRIM21 was an E3 ubiquitin ligase of MFF and promoted its ubiquitination degradation. (A and B) The levels of the remained MFF protein in 40 μg/mL CIP-exposed HTR-8/SVneo cells and with 10 μM CHX treatment within 10 h and its relative quantification (n = 3 independent experiments, Student's t-test). (C) The protein levels of MFF in 40 μg/mL CIP-exposed HTR-8/SVneo cells treated with 10 μM MG132 or 50 μM CQ and co-treated with 10 μM CHX and its relative quantification (n = 3 independent experiments, one-way ANOVA). (D and E) IP assay analysis of the protein levels of MFF-Ub pulled down by MFF in 40 μg/mL CIP-exposed HTR-8/SVneo cells and its relative quantification (n = 3 independent experiments, Student's t-test). (F) MFF IP-MS assay analysis of the proteins that were pulled down by MFF in HTR-8/SVneo cells. (G–K) IP assay analysis of the protein levels of TRIM21 and MFF-Ub pulled down by MFF in HTR-8/SVneo cells with overexpression (Student's t-test) or knockdown (one-way ANOVA) of TRIM21, with MFF and TRIM21 protein levels in cell lysates and their relative quantification (n = 3 independent experiments).

Journal: eBioMedicine

Article Title: Environmental ciprofloxacin triggers pregnancy loss: senescence-driven miscarriage via TRIM21-mediated MFF degradation

doi: 10.1016/j.ebiom.2026.106146

Figure Lengend Snippet: TRIM21 was an E3 ubiquitin ligase of MFF and promoted its ubiquitination degradation. (A and B) The levels of the remained MFF protein in 40 μg/mL CIP-exposed HTR-8/SVneo cells and with 10 μM CHX treatment within 10 h and its relative quantification (n = 3 independent experiments, Student's t-test). (C) The protein levels of MFF in 40 μg/mL CIP-exposed HTR-8/SVneo cells treated with 10 μM MG132 or 50 μM CQ and co-treated with 10 μM CHX and its relative quantification (n = 3 independent experiments, one-way ANOVA). (D and E) IP assay analysis of the protein levels of MFF-Ub pulled down by MFF in 40 μg/mL CIP-exposed HTR-8/SVneo cells and its relative quantification (n = 3 independent experiments, Student's t-test). (F) MFF IP-MS assay analysis of the proteins that were pulled down by MFF in HTR-8/SVneo cells. (G–K) IP assay analysis of the protein levels of TRIM21 and MFF-Ub pulled down by MFF in HTR-8/SVneo cells with overexpression (Student's t-test) or knockdown (one-way ANOVA) of TRIM21, with MFF and TRIM21 protein levels in cell lysates and their relative quantification (n = 3 independent experiments).

Article Snippet: The lysates were incubated with MFF antibody (17090-1-AP, proteintech, dilution 1:200, RRID: AB_2142463 ) or TRIM21 antibody (67136-1-Ig, proteintech, dilution 1:200, RRID: AB_2882435 ) at 4 °C overnight, with equal weight of IgG antibody (ab172730, Abcam, dilution 1:200, RRID: AB_2687931 ) as negative control, and then incubated with Protein A/G magnetic beads (HY–K0202, MedChemExpress) for another 6 h to form bead-immunoprecipitate complex.

Techniques: Ubiquitin Proteomics, Quantitative Proteomics, Protein-Protein interactions, Over Expression, Knockdown

The joint roles of CIP and TRIM21 in MFF ubiquitination degradation. (A) TRIM21 mRNA levels in 0, 1, 10, 20, or 40 μg/mL CIP-exposed HTR-8/SVneo cells (n = 3 independent experiments, one-way ANOVA). (B) TRIM21 protein levels in 0, 1, 10, 20, or 40 μg/mL CIP-exposed HTR-8/SVneo cells and its relative quantification (n = 3 independent experiments, one-way ANOVA). (C and D) IP assay analysis of the protein levels of MFF pulled down by TRIM21 in 40 μg/mL CIP-exposed HTR-8/SVneo cells with MFF and TRIM21 protein levels in cell lysates and its relative quantification (n = 3 independent experiments, Student's t-test). (E–I) IP assay analysis of the protein levels of TRIM21 and MFF-Ub pulled down by MFF in 40 μg/mL CIP-exposed HTR-8/SVneo cells with TRIM21 knockdown, with MFF and TRIM21 protein levels in cell lysates and their relative quantification (n = 3 independent experiments, one-way ANOVA).

Journal: eBioMedicine

Article Title: Environmental ciprofloxacin triggers pregnancy loss: senescence-driven miscarriage via TRIM21-mediated MFF degradation

doi: 10.1016/j.ebiom.2026.106146

Figure Lengend Snippet: The joint roles of CIP and TRIM21 in MFF ubiquitination degradation. (A) TRIM21 mRNA levels in 0, 1, 10, 20, or 40 μg/mL CIP-exposed HTR-8/SVneo cells (n = 3 independent experiments, one-way ANOVA). (B) TRIM21 protein levels in 0, 1, 10, 20, or 40 μg/mL CIP-exposed HTR-8/SVneo cells and its relative quantification (n = 3 independent experiments, one-way ANOVA). (C and D) IP assay analysis of the protein levels of MFF pulled down by TRIM21 in 40 μg/mL CIP-exposed HTR-8/SVneo cells with MFF and TRIM21 protein levels in cell lysates and its relative quantification (n = 3 independent experiments, Student's t-test). (E–I) IP assay analysis of the protein levels of TRIM21 and MFF-Ub pulled down by MFF in 40 μg/mL CIP-exposed HTR-8/SVneo cells with TRIM21 knockdown, with MFF and TRIM21 protein levels in cell lysates and their relative quantification (n = 3 independent experiments, one-way ANOVA).

Techniques: Ubiquitin Proteomics, Quantitative Proteomics, Knockdown

TRIM21 was involved in CIP-caused mitochondrial dysfunctions and senescence in trophoblast cells. (A) CCK8 assay analysis of cell viability in 40 μg/mL CIP-exposed HTR-8/SVneo cells with TRIM21 knockdown for 0, 12, 24, 36, or 48 h (n = 3 independent experiments, one-way ANOVA). (B and C) SA-β-Galactose staining of 40 μg/mL CIP-exposed HTR-8/SVneo cells with TRIM21 knockdown and the quantification of SA-β-Galactose positive cells (n = 3 independent experiments, one-way ANOVA). (D and E) The protein levels of p16, p21, p38, and β-gal in 40 μg/mL CIP-exposed HTR-8/SVneo cells with TRIM21 knockdown and their relative quantification (n = 3 independent experiments, one-way ANOVA). (F) The relative mtDNA copy number in 40 μg/mL CIP-exposed HTR-8/SVneo cells with TRIM21 knockdown (n = 3 independent experiments, one-way ANOVA). (G and H) The relative NAD + /NADH ratios in total lysates (G) or mitochondria (H) of 40 μg/mL CIP-exposed HTR-8/SVneo cells with TRIM21 knockdown (n = 3 independent experiments, one-way ANOVA). (I) The relative quantification of MMP levels in 40 μg/mL CIP-exposed HTR-8/SVneo cells with TRIM21 knockdown (n = 3 independent experiments, one-way ANOVA). (J and K) The representative image of ROS fluorescent signal in 40 μg/mL CIP-exposed HTR-8/SVneo cells with TRIM21 knockdown and the relative quantification of ROS levels (n = 3 independent experiments, one-way ANOVA). (L) The relative quantification of ROS levels in mitochondria of 40 μg/mL CIP-exposed HTR-8/SVneo cells with TRIM21 knockdown (n = 3 independent experiments, one-way ANOVA).

Journal: eBioMedicine

Article Title: Environmental ciprofloxacin triggers pregnancy loss: senescence-driven miscarriage via TRIM21-mediated MFF degradation

doi: 10.1016/j.ebiom.2026.106146

Figure Lengend Snippet: TRIM21 was involved in CIP-caused mitochondrial dysfunctions and senescence in trophoblast cells. (A) CCK8 assay analysis of cell viability in 40 μg/mL CIP-exposed HTR-8/SVneo cells with TRIM21 knockdown for 0, 12, 24, 36, or 48 h (n = 3 independent experiments, one-way ANOVA). (B and C) SA-β-Galactose staining of 40 μg/mL CIP-exposed HTR-8/SVneo cells with TRIM21 knockdown and the quantification of SA-β-Galactose positive cells (n = 3 independent experiments, one-way ANOVA). (D and E) The protein levels of p16, p21, p38, and β-gal in 40 μg/mL CIP-exposed HTR-8/SVneo cells with TRIM21 knockdown and their relative quantification (n = 3 independent experiments, one-way ANOVA). (F) The relative mtDNA copy number in 40 μg/mL CIP-exposed HTR-8/SVneo cells with TRIM21 knockdown (n = 3 independent experiments, one-way ANOVA). (G and H) The relative NAD + /NADH ratios in total lysates (G) or mitochondria (H) of 40 μg/mL CIP-exposed HTR-8/SVneo cells with TRIM21 knockdown (n = 3 independent experiments, one-way ANOVA). (I) The relative quantification of MMP levels in 40 μg/mL CIP-exposed HTR-8/SVneo cells with TRIM21 knockdown (n = 3 independent experiments, one-way ANOVA). (J and K) The representative image of ROS fluorescent signal in 40 μg/mL CIP-exposed HTR-8/SVneo cells with TRIM21 knockdown and the relative quantification of ROS levels (n = 3 independent experiments, one-way ANOVA). (L) The relative quantification of ROS levels in mitochondria of 40 μg/mL CIP-exposed HTR-8/SVneo cells with TRIM21 knockdown (n = 3 independent experiments, one-way ANOVA).

Techniques: CCK-8 Assay, Knockdown, Staining, Quantitative Proteomics

Verification of TRIM21/MFF signalling in human villous tissues. (A and B) TRIM21 protein levels in HC and UM villous tissues and its relative quantification (n = 12, Student's t-test). (C–G) IP assay analysis of the protein levels of TRIM21 and MFF-Ub that were pulled down by MFF in HC and UM villous tissues, with MFF and TRIM21 protein levels in tissue lysates and their relative quantification (each n = 12, Student's t-test). (H) Multivariate logistic regression analysis of forest plot of odds ratio (OR) of TRIM21 protein levels in villous tissues with its 95% confidence interval (95% CI) (n = 12).

Journal: eBioMedicine

Article Title: Environmental ciprofloxacin triggers pregnancy loss: senescence-driven miscarriage via TRIM21-mediated MFF degradation

doi: 10.1016/j.ebiom.2026.106146

Figure Lengend Snippet: Verification of TRIM21/MFF signalling in human villous tissues. (A and B) TRIM21 protein levels in HC and UM villous tissues and its relative quantification (n = 12, Student's t-test). (C–G) IP assay analysis of the protein levels of TRIM21 and MFF-Ub that were pulled down by MFF in HC and UM villous tissues, with MFF and TRIM21 protein levels in tissue lysates and their relative quantification (each n = 12, Student's t-test). (H) Multivariate logistic regression analysis of forest plot of odds ratio (OR) of TRIM21 protein levels in villous tissues with its 95% confidence interval (95% CI) (n = 12).

Techniques: Quantitative Proteomics

Verification of murine Trim21/Mff signalling in CIP-exposed mouse placental tissues. (A) Murine Trim21 mRNA levels in placental tissues of CIP-exposed mice (n = 6, one-way ANOVA). (B and C) Murine Trim21 protein levels in placental tissues of CIP-exposed mice and its relative quantification (n = 6, one-way ANOVA). (D) IP assay analysis of the protein levels of Trim21 and Mff-Ub that were pulled down by Mff in placental tissues of 160 mg/kg/d CIP-exposed mice, with Mff and Trim21 protein levels in tissue lysates (each n = 6). (E) Schematic diagram of CIP-exposed mouse model with Trim21 knockdown. Pregnant mice were treated with saline or 160 mg/kg/d CIP and also treated with 10 mg/kg/3 d AS-Trim 21 (with AS–NC as control). (F and G) Embryo resorption (indicated by red arrows) and the average miscarriage rates in 160 mg/kg/d CIP-exposed mice with Trim21 knockdown (each n = 6, one-way ANOVA). (H) IP assay analysis of the protein levels of Trim21 and Mff-Ub that were pulled down by Mff in 160 mg/kg/d CIP-exposed mice with Trim21 knockdown, with Mff and Trim21 protein levels in tissue lysates (each n = 6). (I) The quantification of positive intensity of SA-β-Galactose staining of placental tissues in 160 mg/kg/d CIP-exposed mice with Trim21 knockdown (each n = 6, one-way ANOVA). (J) The protein levels of p16, p21, p38, and β-gal in placental tissues of 160 mg/kg/d CIP-exposed mice with Trim21 knockdown (each n = 6). (K) The relative mtDNA copy number in placental tissues of 160 mg/kg/d CIP-exposed mice with Trim21 knockdown (n = 6, one-way ANOVA). (L) The NAD + /NADH ratios in placental tissues of 160 mg/kg/d CIP-exposed mice with Trim21 knockdown (n = 6, one-way ANOVA). (M) The MMP levels in placental tissues of 160 mg/kg/d CIP-exposed mice with Trim21 knockdown (n = 6, one-way ANOVA).

Journal: eBioMedicine

Article Title: Environmental ciprofloxacin triggers pregnancy loss: senescence-driven miscarriage via TRIM21-mediated MFF degradation

doi: 10.1016/j.ebiom.2026.106146

Figure Lengend Snippet: Verification of murine Trim21/Mff signalling in CIP-exposed mouse placental tissues. (A) Murine Trim21 mRNA levels in placental tissues of CIP-exposed mice (n = 6, one-way ANOVA). (B and C) Murine Trim21 protein levels in placental tissues of CIP-exposed mice and its relative quantification (n = 6, one-way ANOVA). (D) IP assay analysis of the protein levels of Trim21 and Mff-Ub that were pulled down by Mff in placental tissues of 160 mg/kg/d CIP-exposed mice, with Mff and Trim21 protein levels in tissue lysates (each n = 6). (E) Schematic diagram of CIP-exposed mouse model with Trim21 knockdown. Pregnant mice were treated with saline or 160 mg/kg/d CIP and also treated with 10 mg/kg/3 d AS-Trim 21 (with AS–NC as control). (F and G) Embryo resorption (indicated by red arrows) and the average miscarriage rates in 160 mg/kg/d CIP-exposed mice with Trim21 knockdown (each n = 6, one-way ANOVA). (H) IP assay analysis of the protein levels of Trim21 and Mff-Ub that were pulled down by Mff in 160 mg/kg/d CIP-exposed mice with Trim21 knockdown, with Mff and Trim21 protein levels in tissue lysates (each n = 6). (I) The quantification of positive intensity of SA-β-Galactose staining of placental tissues in 160 mg/kg/d CIP-exposed mice with Trim21 knockdown (each n = 6, one-way ANOVA). (J) The protein levels of p16, p21, p38, and β-gal in placental tissues of 160 mg/kg/d CIP-exposed mice with Trim21 knockdown (each n = 6). (K) The relative mtDNA copy number in placental tissues of 160 mg/kg/d CIP-exposed mice with Trim21 knockdown (n = 6, one-way ANOVA). (L) The NAD + /NADH ratios in placental tissues of 160 mg/kg/d CIP-exposed mice with Trim21 knockdown (n = 6, one-way ANOVA). (M) The MMP levels in placental tissues of 160 mg/kg/d CIP-exposed mice with Trim21 knockdown (n = 6, one-way ANOVA).

Techniques: Quantitative Proteomics, Knockdown, Saline, Control, Staining

Miscarriage treatment. (A) Schematic diagram of CIP-exposed mouse model with F + Q treatment. Pregnant mice were treated with saline or 160 mg/kg/d CIP and also treated with 50 mg/kg/2 d F and 10 mg/kg/2 d Q. (B–D) The protein levels of Trim21 and Mff in placental tissues of 160 mg/kg/d CIP-exposed mice with F + Q treatment and their relative quantification (each n = 6, Student's t-test). (E and F) Embryo resorption (indicated by red arrows) and the average miscarriage rates in 160 mg/kg/d CIP-exposed mice with F + Q treatment (each n = 6, Student's t-test). (G) IP assay analysis of the protein levels of Trim21 and Mff-Ub that were pulled down by Mff in 160 mg/kg/d CIP-exposed mice with F + Q treatment, with Mff and Trim21 protein levels in tissue lysates (each n = 6). (H) The quantification of positive intensity of SA-β-Galactose staining of placental tissues of 160 mg/kg/d CIP-exposed mice with F + Q treatment (each n = 6, Student's t-test). (I) The protein levels of p16, p21, p38, and β-gal in placental tissues of 160 mg/kg/d CIP-exposed mice with F + Q treatment (each n = 6). (J) The relative mtDNA copy number in placental tissues of 160 mg/kg/d CIP-exposed mice with F + Q treatment (n = 6, Student's t-test). (K) The NAD + /NADH ratios in placental tissues of 160 mg/kg/d CIP-exposed mice with F + Q treatment (n = 6, Student's t-test). (L) The MMP levels in placental tissues of 160 mg/kg/d CIP-exposed mice with F + Q treatment (n = 6, Student's t-test). (M) The proposed mechanisms.

Journal: eBioMedicine

Article Title: Environmental ciprofloxacin triggers pregnancy loss: senescence-driven miscarriage via TRIM21-mediated MFF degradation

doi: 10.1016/j.ebiom.2026.106146

Figure Lengend Snippet: Miscarriage treatment. (A) Schematic diagram of CIP-exposed mouse model with F + Q treatment. Pregnant mice were treated with saline or 160 mg/kg/d CIP and also treated with 50 mg/kg/2 d F and 10 mg/kg/2 d Q. (B–D) The protein levels of Trim21 and Mff in placental tissues of 160 mg/kg/d CIP-exposed mice with F + Q treatment and their relative quantification (each n = 6, Student's t-test). (E and F) Embryo resorption (indicated by red arrows) and the average miscarriage rates in 160 mg/kg/d CIP-exposed mice with F + Q treatment (each n = 6, Student's t-test). (G) IP assay analysis of the protein levels of Trim21 and Mff-Ub that were pulled down by Mff in 160 mg/kg/d CIP-exposed mice with F + Q treatment, with Mff and Trim21 protein levels in tissue lysates (each n = 6). (H) The quantification of positive intensity of SA-β-Galactose staining of placental tissues of 160 mg/kg/d CIP-exposed mice with F + Q treatment (each n = 6, Student's t-test). (I) The protein levels of p16, p21, p38, and β-gal in placental tissues of 160 mg/kg/d CIP-exposed mice with F + Q treatment (each n = 6). (J) The relative mtDNA copy number in placental tissues of 160 mg/kg/d CIP-exposed mice with F + Q treatment (n = 6, Student's t-test). (K) The NAD + /NADH ratios in placental tissues of 160 mg/kg/d CIP-exposed mice with F + Q treatment (n = 6, Student's t-test). (L) The MMP levels in placental tissues of 160 mg/kg/d CIP-exposed mice with F + Q treatment (n = 6, Student's t-test). (M) The proposed mechanisms.

Techniques: Saline, Quantitative Proteomics, Staining

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