col2a1 cre mice  (Jackson Laboratory)

Journal: Bone Research

Article Title: Synovial inflammatory macrophage-derived extracellular vesicles exacerbate cartilage lesions with a FMRP-selectively sorted manner in osteoarthritis

doi: 10.1038/s41413-025-00502-4

Figure Lengend Snippet: Synovial pro-inflammatory macrophages-derived EVs aggravate cartilage damage in OA progression. a Representative images of COL2A1 and MMP13 were assessed by IF staining in C28/I2 cells treated with conditioned medium from THP-1 cells. Scale bar: 20 μm. DAPI: 4,6-diamidino-2-phenylindole. b Representative images of COL2A1 and MMP13 were assessed by IF staining in C28/I2 cells treated with EVs from THP-1 cells. Scale bar: 20 μm. DAPI: 4,6-diamidino-2-phenylindole. c Representative transmission electronic microscope (TEM) images of EVs derived from M1-polarized BMDMs in rat models. Scale bar: 100 nm. d A schematic diagram illustrating the experimental design. e The representative confocal images of DiO-labeled EVs derived from M1-polarized BMDMs in tibial cartilage in OA rats at 1 week after surgery. Scale bar: 100 μm. f SO & FG staining in keen joints of sham or OA rats treated with PBS or EVs M1 (up), scale bar: 500 μm. Higher magnification images show dramatic articular cartilage changes (down), scale bar: 100 μm. (left) Quantification of OARSI score was performed using histological sections. n = 7 for each group. (right) IF staining of knee joint sections showing expression of COL2A1 ( g ) and MMP13 ( h ) in articular cartilage. Scale bar: 100 μm. DAPI: 4,6-diamidino-2-phenylindole. Quantification of COL2A1 ( i ) and MMP13 ( j ) expression in articular cartilage with IF staining. n = 7 for each group. One-way ANOVA &Tukey HSD post hoc test (normal distribution) and Kruskal-Wallis Test & Dunn’s test (non-normal distribution) were used for multiple comparisons. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.000 1, ns not significant

Article Snippet: The C57BL/6J- Col2a1 Cre mice were acquired from Cyagen Biosciences Inc (Suzhou, China, No. C001474).

Techniques: Derivative Assay, Staining, Transmission Assay, Microscopy, Labeling, Expressing

Journal: Bone Research

Article Title: Synovial inflammatory macrophage-derived extracellular vesicles exacerbate cartilage lesions with a FMRP-selectively sorted manner in osteoarthritis

doi: 10.1038/s41413-025-00502-4

Figure Lengend Snippet: Synovial pro-inflammatory macrophages-derived EVs miR-155-5p regulates autophagy function of chondrocytes. a Heatmap showing the hierarchical cluster of differential miRNA enrichments in EVs derived from M0 and M1-polarized BMDMs sorted from CD45 + , CD11b + monocytes between sham and OA rats models. n = 2 for each group. b A schematic diagram illustrating the experimental design. c IF staining of knee joint sections showing expression of COL2A1 and MMP13 in articular cartilage. Scale bar: 100 μm. DAPI: 4,6-diamidino-2-phenylindole. d SO & FG staining in keen joints of sham or OA rats treated with PBS, EVs M1 , antagomiR-155-5p, and antagomiR-NC (up), scale bar: 500 μm. Higher magnification images show dramatic articular cartilage changes (down), scale bar: 100 μm. e Transmission electron microscopy (TEM) image of autophagic vesicles in articular cartilage of OA rats. The black arrow indicates the cell bilayer membrane structure of autophagic vesicles. Scale bar: 1 μm. f Quantification of OARSI score was performed using histological sections. n = 7 for each group. g Quantification of autophagic vesicles in articular cartilage of OA rats with TEM detection. n = 7 for each group. One-way ANOVA &Tukey HSD post hoc test (normal distribution) and Kruskal-Wallis Test & Dunn’s test (non-normal distribution) were used for multiple comparisons. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.000 1, ns not significant

Article Snippet: The C57BL/6J- Col2a1 Cre mice were acquired from Cyagen Biosciences Inc (Suzhou, China, No. C001474).

Techniques: Derivative Assay, Staining, Expressing, Transmission Assay, Electron Microscopy, Membrane

Journal: Bone Research

Article Title: Synovial inflammatory macrophage-derived extracellular vesicles exacerbate cartilage lesions with a FMRP-selectively sorted manner in osteoarthritis

doi: 10.1038/s41413-025-00502-4

Figure Lengend Snippet: Synovial pro-inflammatory macrophages-derived EVs miR-155-5p targets GSK-3β/mTORC1 axis in OA chondrocytes. a The online software (TargetScan, Starbase, miRTarget, and PicTar) was used to predict the potential downstream targets of miR-155-5p. b Schematic representation of a predicted binding site of miR-155-5p in the 3’UTR of GSK-3β mRNA, and the mutant GSK-3β 3’UTR. c The luciferase activity was determined using the dual-luciferase reporter system. n = 6 for each group. Representative images ( d ) and quantitative analysis ( h ) of GSK-3β were assessed by IF staining in articular cartilage of sham or OA rats treated with PBS, EVs M1 , antagomiR-155-5p and antagomiR-NC. Scale bar: 100 μm. DAPI: 4,6-diamidino-2-phenylindole. n = 7 for each group. Representative images ( e ) and quantitative analysis of COL2A1 ( i ) and MMP13 ( j ) were assessed by IF staining in GSK-3β KD /SCR KD C28/I2 cells treated with EVs from miR-155-5p KD /miR-NC KD THP-1 cell lines. Scale bar: 20 μm. n = 4 for each group. Representative images ( f ) and quantitative analysis ( k ) of LC3B dots per cell in GSK-3β KD /SCR KD C28/I2 cells treated with EVs from miR-155-5p KD /miR-NC KD THP-1 cell lines. Scale bar: 10 μm. n = 4 for each group. g Western blot showing the mTORC1 signaling pathway-related proteins, including: GSK-3β, p70 S6, P-p70 S6, S6, and P-S6 in GSK-3β KD /SCR KD C28/I2 cells treated with EVs from miR-155-5p KD /miR-NC KD THP-1 cell lines. One-way ANOVA &Tukey HSD post hoc test (normal distribution) and Kruskal-Wallis Test & Dunn’s test (non-normal distribution) were used for multiple comparisons. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.000 1, ns not significant

Article Snippet: The C57BL/6J- Col2a1 Cre mice were acquired from Cyagen Biosciences Inc (Suzhou, China, No. C001474).

Techniques: Derivative Assay, Software, Binding Assay, Mutagenesis, Luciferase, Activity Assay, Staining, Western Blot

Journal: Bone Research

Article Title: Synovial inflammatory macrophage-derived extracellular vesicles exacerbate cartilage lesions with a FMRP-selectively sorted manner in osteoarthritis

doi: 10.1038/s41413-025-00502-4

Figure Lengend Snippet: FMRP selectively loads miR-155-5p into EVs in synovial pro-inflammatory macrophages. a EVs were isolated from plasma of sham rats and OA rats by differential centrifugation, lysed in RIPA buffer. The protein levels of FMRP in EVs were analyzed by Western blot. Ponceau stain is shown as a loading control. b Western blot showing the protein levels of FMRP in plasma EVs isolated from patients with OA or healthy controls. c Enrichments of miR-155-5p in EVs derived from M0 and M1-polarized FMRP OE /Vector OE THP-1 cell lines determined by qRT-PCR. d Enrichments of miR-155-5p in EVs derived from M0 and M1-polarized FMRP KD /SCR KD THP-1 cell lines determined by qRT-PCR. Representative images ( e ) and quantitative analysis of COL2A1 ( g ) and MMP13 ( h ) were assessed by IF staining in C28/I2 cells treated with EVs derived from M0 and M1-polarized FMRP OE /Vector OE /FMRP KD /SCR KD THP-1 cell lines. Scale bar: 20 μm. n = 4 for each group. Representative images ( f ) and quantitative analysis ( i ) of LC3 dots per cell in C28/I2 cells treated with EVs derived from M0 and M1-polarized FMRP OE /Vector OE /FMRP KD /SCR KD THP-1 cell lines. Scale bar: 10 μm. n = 4 for each group. Two-tailed Student’s t test (normal distribution) and Mann-Whitney U test (non-normal distribution) were used for comparisons between the two groups. One-way ANOVA &Tukey HSD post hoc test (normal distribution), Kruskal-Wallis Test & Dunn’s test (non-normal distribution) or Two-way ANOVA & Bonferroni test were used for multiple comparisons. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.000 1, ns not significant

Article Snippet: The C57BL/6J- Col2a1 Cre mice were acquired from Cyagen Biosciences Inc (Suzhou, China, No. C001474).

Techniques: Isolation, Clinical Proteomics, Centrifugation, Western Blot, Staining, Control, Derivative Assay, Plasmid Preparation, Quantitative RT-PCR, Two Tailed Test, MANN-WHITNEY

Journal: Bone Research

Article Title: Synovial inflammatory macrophage-derived extracellular vesicles exacerbate cartilage lesions with a FMRP-selectively sorted manner in osteoarthritis

doi: 10.1038/s41413-025-00502-4

Figure Lengend Snippet: Genetic knockout of miR-155-5p in synovial macrophages retards OA progression. a Breeding strategy of miR-155 CKO mice. b A schematic diagram illustrating the experimental design. c SO & FG staining for keen joints in Control and CKO mice with DMM surgery (up), scale bar: 200 μm. Higher magnification images show dramatic articular cartilage changes (down), scale bar: 50 μm. (left) Quantification of OARSI score was performed using histological sections. n = 7 for each group. (right) d , Representative images (left) and quantitative analysis (right) of HE staining for the synovium in Control and CKO mice with DMM surgery, scale bar: 200 μm. n = 7 for each group. IHC staining of knee joint sections showing expression of COL2A1 ( e ) and MMP13 ( f ) in articular cartilage (up), scale bar: 200 μm. Higher magnification images show dramatic articular cartilage changes (down), scale bar: 50 μm. Quantification of expression of COL2A1 ( g ) and MMP13 ( h ) in articular cartilage of OA mice with IHC staining. n = 7 for each group. i Representative images of LC3 dots per cell in C28/I2 Cells treated with EVs derived from M0 and M1-polarized macrophages in miR-155-5p/miR-NC knockdown (KD) THP-1 cell lines and normal THP-1 cell (Control). n = 4 for each group. Scale bar: 10 μm. Two-tailed Student’s t test (normal distribution) and Mann-Whitney U test (non-normal distribution) were used for comparisons between the two groups. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.000 1, ns not significant

Article Snippet: The C57BL/6J- Col2a1 Cre mice were acquired from Cyagen Biosciences Inc (Suzhou, China, No. C001474).

Techniques: Knock-Out, Staining, Control, Immunohistochemistry, Expressing, Derivative Assay, Knockdown, Two Tailed Test, MANN-WHITNEY

Journal: Bone Research

Article Title: Synovial inflammatory macrophage-derived extracellular vesicles exacerbate cartilage lesions with a FMRP-selectively sorted manner in osteoarthritis

doi: 10.1038/s41413-025-00502-4

Figure Lengend Snippet: Engineering ADSCs-derived EVs promote cartilage repairment in OA model. a Scheme showing the design of engineering ADSCs-derived EVs. b Schematic illustration of the animal experimental procedure. EVs ADSCs or MAP-EVs ADSCs loading with FAM-antagomiR-155-5p were intra-articular injected into OA rats, and the joint samples were collected after 12 h. c IF staining of synovial pro-inflammatory macrophages and fluorescent images of synovium tissues treated with EVs ADSCs or MAP-EVs ADSCs loading with FAM-antagomiR-155-5p. scale bar: 100 μm. d A schematic diagram to evaluate the therapeutic efficacy of engineering EVs ADSCs in OA rats model. Representative images ( e ) and quantitative analysis ( f ) of SO & FG staining in keen joints of sham or OA rats treated with PBS, EVs ADSCs , EVs ADSCs -antagomiR-155-5p and MAP-EVs ADSCs -antagomiR-155-5p. (up), scale bar: 500 μm. Higher magnification images show dramatic articular cartilage changes (down), scale bar: 100 μm. n = 7 for each group. Representative images ( g ) and quantitative analysis ( j ) of IF staining of knee joint sections showing expression of COL2A1 in articular cartilage of the aforementioned rats treated with engineering EVs. Scale bar: 100 μm. DAPI: 4,6-diamidino-2-phenylindole. n = 7 for each group. Representative images ( h ) and quantitative analysis ( k ) of IHC staining of knee joint sections showing expression of MMP13 in articular cartilage of the aforementioned rats treated with engineering EVs. Scale bar: 100 μm. n = 6 for each group. Representative images ( i ) and quantitative analysis ( l ) of IHC staining of knee joint sections showing expression of GSK-3β in articular cartilage of the aforementioned rats treated with engineering EVs. Scale bar: 100 μm. n = 6 for each group. One-way ANOVA &Tukey HSD post hoc test (normal distribution) and Kruskal-Wallis Test & Dunn’s test (non-normal distribution) were used for multiple comparisons. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.000 1, ns not significant

Article Snippet: The C57BL/6J- Col2a1 Cre mice were acquired from Cyagen Biosciences Inc (Suzhou, China, No. C001474).

Techniques: Derivative Assay, Injection, Staining, Drug discovery, Expressing, Immunohistochemistry

Journal: Bone & Joint Research

Article Title: G-protein-coupled estrogen receptor-1 facilitates chondrocyte proliferation in pubertal epiphyseal growth plate via PTHrP/Ihh regulation

doi: 10.1302/2046-3758.147.BJR-2024-0347.R1

Figure Lengend Snippet: The impact of chondrocyte-specific G-protein-coupled estrogen receptor-1 (GPER-1) deficiency on the development of the tibial growth plate in male mice using a GPER-1 conditional knockout ( Col2a1‐Cre; GPER-1 f/f , CKO) model (×400). a) Genotyping of GPER-1 and Col2a1-Cre in the control and CKO mice. b) Representative microslides of the tibial growth plate in four-week-old mice stained and analyzed for GPER-1 and type II collagen (Col-II) expression via immunohistochemistry (IHC). c) Representative microslides and quantification of thicknesses in the resting zone (RZ), proliferative zone (PZ), and hyperopic zone (HZ) via Safranin-O staining. d) Representative microslides and quantification of proliferative chondrocytes stained via Ki67-IHC. e) Representative microslides and quantification of the hyperopic zone stained via type X collagen-IHC. Representative microslides and quantification of f) parathyroid hormone-related peptide (PTHrP)- and g) Indian hedgehog (Ihh)-labelled chondrocytes via IHC staining. h) Ratio of PTHrP-to Ihh-labelled growth plate chondrocyte number. N = 5 for each group. *p < 0.05, **p < 0.01, ***p < 0.001 compared with the control group.

Article Snippet: CKO mice were bred at the National Laboratory Animal Centre (NLAC, NARLabs, Taiwan) through the breeding of GPER-1 tm1c mice (KOMP, University of California, Davis, USA) and Col2a1 ‐Cre mice (JAX stock #003554; Bar Harbor, USA), as described previously.

Techniques: Knock-Out, Control, Staining, Expressing, Immunohistochemistry

Journal: Bone & Joint Research

Article Title: G-protein-coupled estrogen receptor-1 facilitates chondrocyte proliferation in pubertal epiphyseal growth plate via PTHrP/Ihh regulation

doi: 10.1302/2046-3758.147.BJR-2024-0347.R1

Figure Lengend Snippet: Effects of a G-protein-coupled estrogen receptor-1 (GPER-1) agonist (G1) on chondrocyte proliferation and differentiation in micromass-3D cultures. a) Bone mesenchymal stem cells (BMSCs) (D1 cell line) were pelleted into micromasses on day 1 using a chondrogenesis induction medium (×400). Representative microslides of b) Ki67 and c) type II collagen immunofluorescence-stained micromasses on day 7 post-G1 treatment. d) Representative microslides of type X collagen immunofluorescence-stained micromasses on day 14 post-G1 treatment. e) The Ki67-positive cells were quantified as proliferative cells in micromasses, and the results were normalized to those of 4',6-diamidino-2-phenylindole (DAPI) staining. Gene expression levels of f) GPER-1, g) type II collagen, and h) type X collagen in micromasses were analyzed on days 1, 7, and 14 post-G1 treatment. i) CF 594-conjugated anti-parathyroid hormone-related peptide (PTHrP) (red) and CF 488-conjugated anti-Ihh (green) immunofluorescence-stained micromasses on day 14 post-G1 treatment. Intensity of j) PTHrP and k) Indian hedgehog (Ihh) labelling and l) the ratio of PTHrP to Ihh. Gene expression levels of m) PTHrP and n) Ihh in micromasses were analyzed on days 1, 7, and 14 post-G1 treatment. N = 3 for each group. DAPI was used for nuclear staining, and phalloidin was used for actin staining. *p < 0.05, **p < 0.01 compared with the control group.

Article Snippet: CKO mice were bred at the National Laboratory Animal Centre (NLAC, NARLabs, Taiwan) through the breeding of GPER-1 tm1c mice (KOMP, University of California, Davis, USA) and Col2a1 ‐Cre mice (JAX stock #003554; Bar Harbor, USA), as described previously.

Techniques: Immunofluorescence, Staining, Gene Expression, Control

Journal: Cell reports

Article Title: TRIM28 secures skeletal stem cell fate during skeletogenesis by silencing neural gene expression and repressing GREM1/AKT/mTOR signaling axis

doi: 10.1016/j.celrep.2023.112012

Figure Lengend Snippet: (A) Skeletal phenotypes of WT and Trim28 MKO mice at E19.5 visualized with Alcian blue and Alizarin red staining; arrowheads indicate thicker and shorter ribs and over-expanded hindlimb cartilage. Scale bar, 0.5 cm. (B) E17.5 mouse proximal tibial sections stained with Alcian blue Hematoxylin/Orange G. Gross image (left) and magnified views of each zone (right) are shown(RZ, resting zone; PZ, proliferating zone; HZ, hypertrophic zone). Scale bar, 200 μm. (C) Increased cell proliferation in the Trim 28 MKO proximal tibial sections (E17.5) as indicated by Ki67 immunohistochemistry (IHC). Bottom panel: area boxed in red is the magnified perichondrium (PC), while the yellow boxed area is the magnified periarticular resting zone (PRZ). Scale bars, 200 μm. (D) Type II collagen IHC of proximal tibial sections (E17.5). Area boxed in red is magnified in the bottom panels showing high levels of type II collagen retention inthe Trim28 MKO HZ. Scale bars, 200 μm. (E) Type X collagen IHC staining of distal femoral sections (E17.5). The red box area is magnified in the bottom panel, and red dotted lines define type X collagen-expressing areas. Scale bars, 200 μm. See also .

Article Snippet: Trim28 f/f , Dermo1 - Cre and Col2a1 - Cre mice were purchased from Jackson lab (Cat#:018552, Cat#:008712, Cat#: 003554).

Techniques: Staining, Immunohistochemistry, Expressing

Journal: Cell reports

Article Title: TRIM28 secures skeletal stem cell fate during skeletogenesis by silencing neural gene expression and repressing GREM1/AKT/mTOR signaling axis

doi: 10.1016/j.celrep.2023.112012

Figure Lengend Snippet:

Article Snippet: Trim28 f/f , Dermo1 - Cre and Col2a1 - Cre mice were purchased from Jackson lab (Cat#:018552, Cat#:008712, Cat#: 003554).

Techniques: Virus, Recombinant, Plasmid Preparation, SYBR Green Assay, cDNA Synthesis, DNA HS Assay, Reporter Assay, Software, Imaging