plasmid encoding cd63 egfp  (Addgene inc)

Journal: Advanced Science

Article Title: Atrophic Skeletal Muscle‐Derived Extracellular Vesicles Transfer miR‐125a‐5p to Inhibit Bone Formation in Osteoporosis during Aging

doi: 10.1002/advs.202515362

Figure Lengend Snippet: Aged‐SKM‐EVs are increased and taken up by osteoblasts in bone during aging. (A) Representative Hematoxylin and Eosin (H&E) staining of cross‐sections from the mid‐belly of soleus (SOL) muscles and quantification of muscle fiber cross‐sectional area (CSA). n = 6 . Scale bar, 100 µm. (B) Representative tetanic force curve of SOL muscles from 6‐, 12‐, 18‐, and 24‐month‐old mice and quantification of maximal specific force during tetanic stimulation. n = 6 . (C) Representative micro‐CT reconstructed images and quantitative analysis of bone mineral density (BMD), bone volume/total volume (BV/TV), trabecular number (Tb.N), and trabecular separation (Tb.Sp) at the distal femoral metaphysis. Scale bar, 100 µm (upper panels) and 500 µm (lower panels). n = 6 . (D) Representative images of double fluorochrome labeling with calcein green and xylenol orange at the distal femoral metaphysis and quantitative bone histomorphometric analysis of mineral apposition rate (MAR). Scale bar, 25 µm. n = 6 . (E) Representative immunohistochemical staining of osteocalcin (OCN) in distal femoral sections and quantification of osteoblast surface per bone surface (Ob.S/BS). Scale bar, 50 µm. n = 6 . (F) Individual correlations between SOL muscle fiber CSA and BMD, and between CSA and Ob.S/BS. n = 24 . (G) Western blot analysis and quantification of protein expression of RAB27A expression in SOL muscles. n = 3 . (H) Representative immunohistochemical staining and quantification of RAB27A‐positive areas in SOL muscles. Scale bar, 100 µm. n = 6 . (I) Representative transmission electron microscopy (TEM) images of SKM‐EVs. Scale bar, 1 µm. (J) Nanoparticle tracking analysis (NTA) showing particle size distribution and concentration of SKM‐EVs. (K) Western blot analysis of canonical EV markers in SKM‐EVs. (L) Distribution and quantification of eGFP‐labeled EVs in skeletal muscle from HSA Cre ;Cd63 ( loxp − eGFP ) mice. Scale bar, 100 µm. n = 3 . (M) Representative fluorescence images showing circulating eGFP + PKH26 + EVs in plasma from HSA Cre ;Cd63 (loxp‐eGFP) mice, and quantification of the number density of eGFP + PKH26 + EVs (per µm 2 ). Scale bar, 100 nm. n = 6 . (N) Representative immunofluorescence staining of OCN in femoral sections (left), quantitative colocalization analysis (middle), and quantification of the percentage of CD63 GFP + OCN + cells among total OCN + cells (right). Nuclei were co‐stained with DAPI. Scale bar, 100 µm. n = 3 . All data are presented as mean ± SEM. P values were determined by one‐way analysis of variance (ANOVA) followed by Tukey's multiple comparisons test (A–E, G–H), or unpaired two‐tailed Student's t test (L–N). * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Cd63 (loxp‐eGFP) mice were obtained from Shanghai Model Organisms, and Dicer fl/fl mice were purchased from GemPharmatech.

Techniques: Staining, Muscles, Micro-CT, Labeling, Immunohistochemical staining, Western Blot, Expressing, Transmission Assay, Electron Microscopy, Concentration Assay, Fluorescence, Clinical Proteomics, Immunofluorescence, Two Tailed Test

Journal: Advanced Science

Article Title: Atrophic Skeletal Muscle‐Derived Extracellular Vesicles Transfer miR‐125a‐5p to Inhibit Bone Formation in Osteoporosis during Aging

doi: 10.1002/advs.202515362

Figure Lengend Snippet: Muscle‐specific inhibition of EV generation alleviates bone loss in aged mice. (A) Schematic diagram illustrating the experimental design. Aged mice were intravenously injected with RGDLTTP peptide‐modified liposome capsulating GW4869 every three days for 6 weeks. (B) Representative immunofluorescent staining for CD63 in TA muscle sections. Nuclei were co‐stained with DAPI. Scale bars, 100 µm. (C) Representative micro‐CT reconstructed images of femora. Scale bar, 100 µm (upper panels) and 500 µm (lower panels). (D) Micro‐CT analysis of BMD, Tb.N, Tb.Th and Tb.Sp at the distal femoral metaphysis. n = 6 . (E) Representative images of double fluorochrome labeling with calcein green and xylenol orange at the distal femoral metaphysis and quantitative bone histomorphometric analysis of MAR. Scale bar, 25 µm. n = 6 . (F) Representative immunohistochemical staining of OCN in distal femoral sections and quantification of Ob.S/BS. Scale bar, 50 µm. n = 6 . (G) Schematic diagram illustrating the experimental design of co‐culture preosteoblasts with control myotubes (CON), atrophic myotubes (ATR) or GW4869 pretreated atrophic myotubes (ATR+GW4869). Myotubes were treated with or without GW4869 (20 µм) before co‐culture. (H) NTA analysis of EVs derived from ATR myotubes treated with DMSO or GW4869. (I) qPCR analysis of osteogenic differentiation‐related genes in preosteoblast after co‐cultured with indicated myotubes. n = 3 . (J,K) Western blot analysis and quantification of osteogenic differentiation‐related protein expression in osteoblasts after co‐cultured with indicated myotubes. n = 3 . (L) Alkaline phosphatase (ALP) staining of osteoblasts after co‐cultured with indicated myotubes for 7 days, respectively. Scale bars, 500um. All data are presented as mean ± SEM. P values were determined by unpaired two‐tailed Student's t test (D‐F), or one‐way ANOVA followed by Tukey's multiple comparisons test (I, K). * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Cd63 (loxp‐eGFP) mice were obtained from Shanghai Model Organisms, and Dicer fl/fl mice were purchased from GemPharmatech.

Techniques: Inhibition, Injection, Modification, Staining, Micro-CT, Labeling, Immunohistochemical staining, Co-Culture Assay, Control, Derivative Assay, Cell Culture, Western Blot, Expressing, Two Tailed Test

Journal: Journal of Extracellular Vesicles

Article Title: Cholesterol Deficiency Directs Autophagy‐Dependent Secretion of Extracellular Vesicles

doi: 10.1002/jev2.70218

Figure Lengend Snippet: sEVs from cholesterol‐depleted cells are more readily internalized. (A) Stably transfected HEK293T cells overexpressing mEmerald‐CD9 or eGFP‐CD63. Scale bar, 200 µm. (B) Experimental design for isolation of mEmerald‐CD9 and eGFP‐CD63 sEVs and quantitation of uptake by recipient cells. (C) Representative 3D reconstructions of target celluptake of mEmerald‐CD9 sEVs. Plasma membrane visualized with concanavalin A (ConA). Scale bar, 25 µm. (D) Representative images of target cells incubated with sEVs from cells treated with vehicle, simvastatin, or AY9944. Scale bar, 25 µm. (E) Quantified uptake in recipient cells exposed to 1 × 10 8 or 2.5 × 10 8 mEmerald‐CD9 sEVs (mean ± SEM; n = 21, 7 images analyzed from 3 independent experiments). Two‐way ANOVA (Interaction effect: F2,120 = 26.31, p < 0.0001; Treatment effect: F2,120 = 70.3, p < 0.0001; Dose effect: F1,120 = 152.1, p < 0.0001). Sidak's multiple comparisons test (** p ≤ 0.005, **** p < 0.0001). (F) Quantified uptake in recipient cells exposed to 1 × 10 8 or 2.5 × 10 8 eGFP‐CD63 sEVs (mean ± SEM; n = 12, 4 images analyzed from 3 independent experiments). Two‐way ANOVA (interaction effect: F2,66 = 5.633, p ≤ 0.0055; Treatment effect: F2,66 = 40.73, p < 0.0001; Dose effect: F1,66 = 58.16, p < 0.0001). Sidak's multiple comparisons test (* p ≤ 0.05, ** p ≤ 0.005, **** p < 0.0001).

Article Snippet: Plasmids housing genes for proteins involved in sEV biogenesis or mature autophagosome formation fused to fluorescent reporters, including mEmerald‐CD9 (a gift from Michael Davidson, Addgene plasmid # 54029), eGFP‐CD63 (a gift from Paul Luzio, Addgene plasmid # 62964) and mCherry‐LC3B (a gift from David Rubinsztein, Addgene plasmid # 40827), were isolated and purified (GeneJET Plasmid Maxiprep Kit, Cat. # K0491).

Techniques: Stable Transfection, Transfection, Isolation, Quantitation Assay, Clinical Proteomics, Membrane, Incubation

Journal: Nature Communications

Article Title: Osteocyte-derived extracellular vesicles mediate the bone-to-cartilage crosstalk and promote osteoarthritis progression

doi: 10.1038/s41467-025-59861-5

Figure Lengend Snippet: A A schematic diagram illustrating the workflow utilizing primary human osteoarthritic chondrocytes co-cultured with osteocytes located in high stress (HS) and low stress (LS) region of the subchondral bone (SCB). B Levels of IL-1β, IL-6 and TNF-α measured by ELISA assays ( n = 6). C The Alcian blue staining. Scale bar, 1 mm. D qPCR analysis of COL2A1, ACAN, SOX9, MMP13 and ADAMTS5 levels in primary human osteoarthritic chondrocytes co-cultured with LS/HS tissues ( n = 3). E Western blot of COLII, ACAN, SOX9, MMP13 and ADAMTS5 protein expression in primary human osteoarthritic chondrocytes co-cultured with LS/HS tissues ( n = 3). F RNA-sequencing and Gene Ontology (GO) enrichment analysis of the genes in LS/HS tissues. G A heat map (top) and its quantitation (bottom) indicating the expression of genes associated with extracellular vesicles (EVs) biogenesis within LS/HS tissues. H A schematic diagram summarizing the biology of EV biogenesis. I Representative electron microscopy images of intraluminal vesicles (ILVs) in LS/HS tissues. Scale bar, 500 nm. J Illustration of the animal experimental protocol for osteoarthritis (OA) modeling using osteocyte-specific ILVs/EVs reporter mice. K Safranin O staining. Scale bar, 200 μm. L The Osteoarthritis Research Society International (OARSI) scores ( n = 6). M Localization of Cd63-GFP (green) in SCB osteocytes. Scale bar, 50 μm. SCB Subchondral bone, BM Bone marrow. N The semi-quantitative analysis of Cd63-GFP + positive cells in SCB osteocytes ( n = 6). O Localization of Cd63-GFP (green) in articular cartilage. The white arrows indicated Cd63-GFP (green). Scale bar, 50 μm. P The semi-quantitative analysis of Cd63-GFP + positive cells in articular cartilage ( n = 6). Data are presented as the mean ± SD. P values are from paired two-tailed Student’s t -test ( B , D ), Kruskal–Wallis test followed by two-tailed Mann–Whitney U test ( L ), one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test ( N , P ) or Hypergeometric test ( F ). Source data are provided as a Source Data file.

Article Snippet: Cd63 (loxp-eGFP) mice were purchased from Shanghai Model Organisms.

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay, Staining, Western Blot, Expressing, RNA Sequencing, Quantitation Assay, Electron Microscopy, Two Tailed Test, MANN-WHITNEY

Journal: Nature Communications

Article Title: Osteocyte-derived extracellular vesicles mediate the bone-to-cartilage crosstalk and promote osteoarthritis progression

doi: 10.1038/s41467-025-59861-5

Figure Lengend Snippet: A A schematic diagram illustrated that primary human osteoarthritic chondrocytes were treated with extracellular vesicles (EVs) extracted from low stress/high stress tissues (LS/HS-EVs). B The nanoparticle tracking analysis (NTA) analysis. C Representative confocal images showing the uptake of LS/HS-EVs in chondrocytes. Scale bar, 30 μm. D Alcian blue staining. Scale bar, 1 mm. E Western blot analysis of COLII, ACAN, SOX9, MMP13 and ADAMTS5 protein expression ( n = 3). F A schematic diagram illustrating the administration of intra-articular injection of LS/HS-EVs to destabilization of the medial meniscus (DMM) mice. G Representative confocal images of PKH26-labeled osteocyte-EVs in the cartilage. Scale bar, 50 μm. H The Safranin O images ( n = 8). Scale bar in upper panel, 200 μm. Scale bar in lower panel, 100 μm. I A schematic diagram illustrated that primary mouse chondrocytes were treated with Static/Load-EVs. J The NTA analysis. K Representative confocal fluorescence micrographs showing the uptake of EVs in chondrocytes. Scale bar, 30 μm. L Alcian blue staining. Scale bar, 2 mm. M Western blot analysis of ColII, Acan, Sox9, Mmp13 and Adamts5 protein level ( n = 3). N A schematic diagram illustrating the administration of intra-articular injection of Static-EVs and Load-EVs to DMM mice. O Representative confocal images of PKH26-labeled MLO-Y4-EVs in the cartilage. Scale bar, 50 μm. P The Safranin O images of cartilage ( n = 8). Scale bar in upper panel, 200 μm. Scale bar in lower panel, 100 μm. Q A schematic diagram illustrating the strategy for reducing osteocyte-derived EVs in DMM mice. R Representative immunohistochemical staining of Cd63 in subchondral bone (SCB) osteocytes ( n = 8). Scale bars: 50 μm. S The Safranin O images ( n = 8). Scale bar in upper panel, 200 μm. Scale bar in lower panel, 100 μm. Data are presented as the mean ± SD. Data was analyzed by unpaired two-tailed Student’s t -test ( R ). Source data are provided as a Source Data file.

Article Snippet: Cd63 (loxp-eGFP) mice were purchased from Shanghai Model Organisms.

Techniques: Staining, Western Blot, Expressing, Injection, Labeling, Fluorescence, Derivative Assay, Immunohistochemical staining, Two Tailed Test