Journal: Journal of Virology

Article Title: Severe Fever with Thrombocytopenia Syndrome Virus NSs Interacts with TRIM21 To Activate the p62-Keap1-Nrf2 Pathway

doi: 10.1128/JVI.01684-19

Figure Lengend Snippet: NSs activates the Nrf2 pathway and induces Nrf2-mediated gene expression. (A and B) (Left) RAW 264.7 cells stably expressing the vector, NSs-WT, or NSs-A46 were harvested, and WCEs (A) and the cytoplasmic/nuclear fractions (B) were analyzed by immunoblotting for the indicated proteins. (Right) The relative levels of Nrf2 in RAW 264.7 cells compared to the levels of β-actin for WCEs (A) and p84 for the nucleus (B) were calculated. (C and D) The mRNA levels of the Nrf2 target genes (Hmox1 and Nqo1) (C) and Nrf2 (D) in RAW 264.7 cells expressing the vector, NSs-WT, or NSs-A46 were measured by qRT-PCR. (E) The Nqo1 mRNA level in RAW 264.7 cells treated with dimethyl sulfoxide (DMSO) and an Nrf2 inhibitor (INH; 10 μM) for 24 h was measured by qRT-PCR.

Article Snippet: The primary antibodies included TRIM21 (clone D1O1D; Cell Signaling), Nrf2 (clone D1Z9C; Cell Signaling), p62 (Cell Signaling), Keap1 (clone D6B12; Cell Signaling), GFP (clone B-2; Santa Cruz), GST (clone B-14; Santa Cruz), β-actin (clone C4; Santa Cruz), Flag (for rabbit Flag, clone F7425 [Sigma]; for mouse Flag, clone F1804 [Sigma]), HA (for rabbit HA, clone PRB-101P [Covance]; for mouse HA, clone 16B12 [BioLegend]), V5 (for rabbit V5, clone A190 [Bethyl]; for mouse V5, Invitrogen), and Myc (for rabbit Myc, clone Poly9063 [BioLegend]; for mouse Myc, clone 9E10 [BioLegend]).

Techniques: Gene Expression, Stable Transfection, Expressing, Plasmid Preparation, Western Blot, Quantitative RT-PCR

Journal: Journal of Virology

Article Title: Severe Fever with Thrombocytopenia Syndrome Virus NSs Interacts with TRIM21 To Activate the p62-Keap1-Nrf2 Pathway

doi: 10.1128/JVI.01684-19

Figure Lengend Snippet: NSs induces CD36 expression via the Nrf2 pathway and increases lipid uptake. (A) The level of Cd36 mRNA in RAW 264.7 cells expressing the vector, NSs-WT, or NSs-A46 was measured by qRT-PCR. (B) CD36 surface expression by RAW 264.7 cells expressing the vector, NSs-WT, or NSs-A46 was measured by flow cytometry. The surface expression of FITC fluorescence on cells was determined using FlowJo software (left), and the relative expression levels are provided (right). FACS, fluorescence-activated cell sorting. (C) The level of Cd36 mRNA in RAW 264.7 cells treated with DMSO or an Nrf2 inhibitor (INH, 10 μM) for 24 h was measured by qRT-PCR. (D) RAW 264.7 cells were treated with rabbit IgG-FITC complexed with latex beads at a final dilution of 1:500. (Right) The cells were washed/harvested, and flow cytometry was applied to measure the internalized rabbit IgG-FITC-complexed latex beads. (Left) The percentage of cells that phagocytosed the beads was determined using FlowJo software. (E) The organic phase of RAW 264.7 cells was extracted to measure the amount of free or total (free and esterified) cholesterol. A colorimetric assay was used to measure intracellular cholesterol levels at an absorbance of 570 nm, based on cholesterol standards. (F) RAW 264.7 cells were plated and treated with fluorescently tagged cholesterol at 20 μg/ml for 48 h. The cells were washed, fixed with paraformaldehyde, and applied to a microplate reader with filter sets designed for FITC and GFP. The amount of cholesterol taken up was described at a relative level.

Article Snippet: The primary antibodies included TRIM21 (clone D1O1D; Cell Signaling), Nrf2 (clone D1Z9C; Cell Signaling), p62 (Cell Signaling), Keap1 (clone D6B12; Cell Signaling), GFP (clone B-2; Santa Cruz), GST (clone B-14; Santa Cruz), β-actin (clone C4; Santa Cruz), Flag (for rabbit Flag, clone F7425 [Sigma]; for mouse Flag, clone F1804 [Sigma]), HA (for rabbit HA, clone PRB-101P [Covance]; for mouse HA, clone 16B12 [BioLegend]), V5 (for rabbit V5, clone A190 [Bethyl]; for mouse V5, Invitrogen), and Myc (for rabbit Myc, clone Poly9063 [BioLegend]; for mouse Myc, clone 9E10 [BioLegend]).

Techniques: Expressing, Plasmid Preparation, Quantitative RT-PCR, Flow Cytometry, Fluorescence, Software, FACS, Colorimetric Assay

Journal: Bone Research

Article Title: Trim21 depletion alleviates bone loss in osteoporosis via activation of YAP1/β-catenin signaling

doi: 10.1038/s41413-023-00296-3

Figure Lengend Snippet: Trim21 is elevated in osteoporotic patients, and its deficiency leads to high bone mass. a Quantitative RT‒PCR analysis of Trim21 mRNA expression in bone specimens from patients with different bone mineral densities (BMDs), which were defined as normal, osteopenia, and osteoporosis. b Correlation analysis between Trim21 mRNA expression and RF-BMD and LS-BMD. RF, right femur; LS, lumbar spine. c Immunoblotting analysis of Trim21 protein expression in the lumbar vertebra of 5-month-old sham-operated or ovariectomized mice. d Schematic diagram showing the analysis of skeletal parameters of mice at different ages. e Alcian blue/Alizarin Red staining of the whole skeleton of 1-week-old Trim21 +/+ , Trim21 +/− , and Trim21 −/− littermates. f X-ray images of Trim21 +/+ and Trim21 −/− mice at 1 month and 6 months (left panel). Quantitative analysis of the tibia length of mice at different ages (right panel). g Representative H&E and S/O staining images of tibial sections from 1-month-old Trim21 +/+ and Trim21 −/− mice (left panel). Quantitative analysis of the growth plate thickness of the indicated mice (right panel). h , i Representative immunofluorescence images ( h ) showing the expression of Sox9 + cells ( i ) in growth plates of tibial sections in 1-month-old Trim21 +/+ and Trim21 −/− mice. j Representative micro-CT images of the proximal tibia bone of 14-week-old mice. Quantitative measurements of bone volume per tissue volume (BV/TV), trabecular thickness (Tb. Th), trabecular number (Tb. N), and trabecular separation (Tb.Sp). All bar graphs are presented as the mean ± SD. * P < 0.05; *** P < 0.001; **** P < 0.000 1; n.s. not significant by Student’s t test

Article Snippet: BMMs were derived from 4- to 8-week-old Trim21 +/+ and Trim21 −/− mice or Trim21 f/f mice, as well as Ctsk-cre; Trim21 f/f mice, and then incubated in α-MEM containing 30 ng·mL −1 M-CSF (MCE, Cat# HY-P70553, USA) for 4 days to generate BMMs.

Techniques: Expressing, Western Blot, Staining, Immunofluorescence, Micro-CT

Journal: Bone Research

Article Title: Trim21 depletion alleviates bone loss in osteoporosis via activation of YAP1/β-catenin signaling

doi: 10.1038/s41413-023-00296-3

Figure Lengend Snippet: Loss of Trim21 enhances osteoblast activity and favors bone formation. a , b Representative immunoblotting analysis ( a ) and quantification of Runx2, Osterix, and Trim21 in MC3T3-E1 cells ( b ) treated with osteogenic medium for 0, 4, and 7 days. c Quantitative RT‒PCR analysis of osteogenic biomarker genes ( Osterix, Runx2, and Trim21 ) in OBs with osteogenic induction. d , e Alizarin Red S (upper panel) and ALP (lower panel) staining of primary osteoblasts (OBs) after induction with osteogenic medium for different times ( d ). The percentage of Alizarin Red S- ( n ≥ 3) and ALP- ( n ≥ 3) stained area ( e ). f Quantitative RT‒PCR detection of osteogenic biomarker genes ( Runx2, Osterix, OCN, OPG, and RANKL ) in OBs derived from Trim21 −/ − and Trim21 +/+ mice upon osteogenic induction for 7 days. g , h Representative immunoblotting analysis ( g ) and quantification of Runx2 and Osterix in OBs ( h ) after osteogenic induction for 0, 4, and 7 days. i Representative micro-CT images of calvarial bone defects in 2-month-old Trim21 +/+ and Trim21 −/ − mice after surgical induction for 1 month (left panel). Quantitative measurements of bone volume per tissue volume (BV/TV) and bone defect diameter (right panel). All bar graphs are presented as the mean ± SD. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.000 1; n.s. not significant by Student’s t test

Article Snippet: BMMs were derived from 4- to 8-week-old Trim21 +/+ and Trim21 −/− mice or Trim21 f/f mice, as well as Ctsk-cre; Trim21 f/f mice, and then incubated in α-MEM containing 30 ng·mL −1 M-CSF (MCE, Cat# HY-P70553, USA) for 4 days to generate BMMs.

Techniques: Activity Assay, Western Blot, Biomarker Discovery, Staining, Derivative Assay, Micro-CT

Journal: Bone Research

Article Title: Trim21 depletion alleviates bone loss in osteoporosis via activation of YAP1/β-catenin signaling

doi: 10.1038/s41413-023-00296-3

Figure Lengend Snippet: Loss of Trim21 inhibits osteoclast formation and differentiation. a Representative image of histological sections of the tibia that were stained with TRAP (left panel). Bone marrow (BM) and trabecular bone (TB) are indicated in black. TRAP-stained osteoclasts (OCs) are denoted by the red arrow. OC. N/BPm (OC number per bone parameter) and OC. S/BS (OC surface per bone surface) were determined (right panel). Scale bar: 100 μm. b Quantification of F-actin ring number in BMM-derived OCs from immunofluorescence staining of Fig. . c Representative immunoblot analysis and quantification of Ctsk expression in BMM-derived OCs from Trim21 +/+ and Trim21 −/− mice. d , e Quantitative RT‒PCR detection of OC differentiation genes ( Ctsk, Nfatc1, Acp5, ATP6vod2, and Mmp9 ) in BMM-derived OCs from Trim21 +/+ and Trim21 − / − mice. PBS indicates PBS containing 30 ng·mL −1 M-CSF, while RANKL indicates induction with 30 ng·mL −1 M-CSF and 100 ng·mL −1 RANKL f , g Quantitative RT‒PCR detection ( f ) of OC differentiation genes ( Ctsk and Nfatc1 ) and TRAP staining ( g ) in BMM-derived OCs from Trim21 f/f mice treated with 30 ng·mL −1 M-CSF plus 100 ng·mL −1 RANKL or PBS containing 30 ng·mL −1 M-CSF for 5 days and infected with lentivirus expressing EGFP or Cre recombinase (defined as LV-Con or LV-Cre, respectively). Quantification of TRAP-positive OCs and the number of nuclei per TRAP + cell (right panel) ( g ). Scale bar: 50 μm. h BMMs derived from 4-week-old Trim21 f/f and Ctsk-cre; Trim21 f/f mice were induced for OC differentiation with either 30 ng·mL −1 M-CSF plus 100 ng·mL −1 RANKL or PBS containing 30 ng·mL −1 M-CSF for 5 days (left panel). Quantification of TRAP-positive OCs and the number of nuclei per TRAP + cell (right panel). Scale bar: 50 μm. i , j Schematic diagram illustrating the coculture model of OCs with BMSCs from Trim21 +/+ and Trim21 − / − mice ( i ). Representative images (left panel) and quantification data of TRAP-positive OCs and nucleus number per TRAP + cell (right panel) ( j ). Scale bar: 50 μm. All bar graphs are presented as the mean ± SD. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.000 1; n.s. not significant by Student’s t test

Article Snippet: BMMs were derived from 4- to 8-week-old Trim21 +/+ and Trim21 −/− mice or Trim21 f/f mice, as well as Ctsk-cre; Trim21 f/f mice, and then incubated in α-MEM containing 30 ng·mL −1 M-CSF (MCE, Cat# HY-P70553, USA) for 4 days to generate BMMs.

Techniques: Staining, Derivative Assay, Immunofluorescence, Western Blot, Expressing, Infection

Journal: Bone Research

Article Title: Trim21 depletion alleviates bone loss in osteoporosis via activation of YAP1/β-catenin signaling

doi: 10.1038/s41413-023-00296-3

Figure Lengend Snippet: YAP1/β-catenin signaling is essential for Trim21-mediated osteogenic differentiation. a Schematic diagram showing TMT-based quantitative proteomics for the identification of differentially expressed proteins (DEPs) in bone marrow mesenchymal stem cells (BMSCs) derived from Trim21 +/+ and Trim21 −/− mice. b Volcano plots of DEPs in BMSCs from Trim21 +/+ and Trim21 −/− mice. c Heatmap analysis of DEPs in BMSCs. Three replicates of each group were included, and the top 29 DEPs are shown. d KEGG enrichment analysis of the DEPs in the BMSCs. e Representative immunoblotting analysis and quantification of BCL9, AXIN1, and YAP1 in BMSCs; proteomics sample: part of the samples subjected to proteomics analysis. f Representative immunoblotting analysis and quantification of BCL9, β-catenin, YAP1, and Runx2 protein expression in BMSCs after osteogenic induction for 7 days. g The endogenous interaction between Trim21, BCL9, β-catenin, and YAP1 was evaluated using a co-IP assay. h Protein‒protein interaction of YAP1 and Trim21 in living cells. The two BiFC plasmids encoding Myc-VN155-YAP1 and HA-VC155-Trim21 along with HA-cerulean were cotransfected into HEK293T cells for 24 h. Representative images showing transfected cells (cerulean) and the interaction between YAP1 and Trim21 (Venus). Nuclei were stained with DAPI. Scale bar: 20 μm. i Immunoblotting analysis of BCL9, β-catenin, YAP1, and HA-Trim21 protein expression in HEK293T cells treated with or without MG132. j Representative images showing the expression of YAP1 + OBs derived from Trim21 +/+ and Trim21 −/ − mice. Scale bar: 20 μm. All bar graphs are presented as the mean ± SD. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.000 1; n.s. not significant by Student’s t test

Article Snippet: BMMs were derived from 4- to 8-week-old Trim21 +/+ and Trim21 −/− mice or Trim21 f/f mice, as well as Ctsk-cre; Trim21 f/f mice, and then incubated in α-MEM containing 30 ng·mL −1 M-CSF (MCE, Cat# HY-P70553, USA) for 4 days to generate BMMs.

Techniques: Quantitative Proteomics, Derivative Assay, Western Blot, Expressing, Co-Immunoprecipitation Assay, Transfection, Staining

Journal: Bone Research

Article Title: Trim21 depletion alleviates bone loss in osteoporosis via activation of YAP1/β-catenin signaling

doi: 10.1038/s41413-023-00296-3

Figure Lengend Snippet: Loss of Trim21 protects mice from lipopolysaccharide (LPS)-induced bone loss. a Quantitative RT‒PCR determination of IL-6 , Osterix , and Runx2 mRNA expression in OBs with or without lipopolysaccharide (LPS) treatment during osteogenic induction. b Schematic diagram showing the H&E staining and micro-CT analysis of Trim21 +/+ and Trim21 − /− mice induced by PBS or LPS. c Representative images of H&E staining of tibia sections of 13-week-old Trim21 +/+ and Trim21 −/− mice induced by PBS or LPS. Bone marrow (BM) and trabecular bone (TB) are labeled with red arrows. d , e Representative micro-CT images ( d ) and BV/TV ( e ) of proximal tibia trabecular bone of 13-week-old Trim21 +/+ and Trim21 −/− mice induced by PBS or LPS. f The bone loss ratio after LPS treatment in global knockout mice (left panel) and conditional knockout mice (right panel) . g Representative micro-CT images and BV/TV of proximal tibia trabecular bone of 13-week-old Trim21 f/f and Ctsk-cre; Trim21 f/f mice induced by PBS or LPS. h Representative micro-CT images of cortical bones and quantification of BV/TV (left panel) and thickness (right panel) of Trim21 f/f and Ctsk-cre; Trim21 f/f mice induced by either PBS or LPS. All bar graphs are presented as the mean ± SD. * P < 0.05; ** P < 0.01; *** P < 0.001; n.s. not significant by Student’s t test

Article Snippet: BMMs were derived from 4- to 8-week-old Trim21 +/+ and Trim21 −/− mice or Trim21 f/f mice, as well as Ctsk-cre; Trim21 f/f mice, and then incubated in α-MEM containing 30 ng·mL −1 M-CSF (MCE, Cat# HY-P70553, USA) for 4 days to generate BMMs.

Techniques: Expressing, Staining, Micro-CT, Labeling, Knock-Out

Journal: Bone Research

Article Title: Trim21 depletion alleviates bone loss in osteoporosis via activation of YAP1/β-catenin signaling

doi: 10.1038/s41413-023-00296-3

Figure Lengend Snippet: Trim21 orchestrates ovariectomy (OVX)-induced bone metabolism by targeting YAP1 signaling. a Determination of fat cell density in the proximal tibia of 20-week-old Trim21 +/+ and Trim21 −/− mice induced by sham operation or OVX. b Representative micro-CT images and quantitative data (BV/TV) of proximal tibial bone of 20-week-old Trim21 +/+ and Trim21 −/− mice induced by sham operation or OVX. c , d Calcein double labeling of mineral layers of tibial trabecular bone of 5-month-old mice. e Representative images of von Kossa staining of the undecalcified proximal tibia of 5-month-old mice. Scale bar: 50 μm. f IHC staining images of the proximal tibia of 5-month-old mice using an antibody against YAP1. The YAP1-stained positive cells are denoted by the red arrow. Scale bar: 50 μm. g , h Representative images of histological sections of the tibia that were stained with TRAP in Trim21 +/+ and Trim21 −/− mice induced by sham operation or OVX. TRAP-stained osteoclasts (OCs) are denoted by the red arrow. OC. N/BPm (OC number per bone parameter) and OC. S/BS (OC surface per bone surface) was determined. Scale bar: 100 μm. All bar graphs are presented as the mean ± SD. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.000 1; n.s. not significant by Student’s t test

Article Snippet: BMMs were derived from 4- to 8-week-old Trim21 +/+ and Trim21 −/− mice or Trim21 f/f mice, as well as Ctsk-cre; Trim21 f/f mice, and then incubated in α-MEM containing 30 ng·mL −1 M-CSF (MCE, Cat# HY-P70553, USA) for 4 days to generate BMMs.

Techniques: Micro-CT, Labeling, Staining, Immunohistochemistry

Journal: Bone Research

Article Title: Trim21 depletion alleviates bone loss in osteoporosis via activation of YAP1/β-catenin signaling

doi: 10.1038/s41413-023-00296-3

Figure Lengend Snippet: A schematic of Trim21 in the regulation of bone remodeling via YAP1/β-catenin signaling. Normal bone remodeling is maintained by the balance of MSC/osteoblast-mediated bone formation and osteoclast-mediated bone resorption. Trim21, by interacting with the protein complex formed by YAP1/β-catenin/BCL9, dictates the degradation of this protein complex, which in turn inactivates YAP1 and β-catenin signaling, which is essential for osteoblast differentiation. However, Trim21 is critical for maintaining the basic expression of osteoclast biomarkers, including Nfatc1 and Ctsk . Therefore, the coupling of osteoblasts with osteoclasts is attributed to dynamic changes in bone metabolism (left panel). In contrast, the loss of Trim21 causes disassociation with the YAP1/β-catenin/BCL9 complex, which then enters the nucleus for subsequent activation of osteogenic genes, including Runx2 and Osterix . In addition, the loss of Trim21 suppresses the maturation of osteoclasts. Together, these results indicate that Trim21 deficiency alleviates pathological bone loss by activating YAP1/β-catenin signaling

Article Snippet: BMMs were derived from 4- to 8-week-old Trim21 +/+ and Trim21 −/− mice or Trim21 f/f mice, as well as Ctsk-cre; Trim21 f/f mice, and then incubated in α-MEM containing 30 ng·mL −1 M-CSF (MCE, Cat# HY-P70553, USA) for 4 days to generate BMMs.

Techniques: Expressing, Activation Assay