Journal: Journal of Molecular Medicine (Berlin, Germany)

Article Title: Targeting EFEMP1 enhances chondrogenesis and inhibits hypertrophic differentiation in a spontaneous osteoarthritis mouse model

doi: 10.1007/s00109-026-02656-y

Figure Lengend Snippet: Effect of EFEMP1 knockdown by small interfering RNA (siRNA) on chondrocyte phenotype. A The human chondrocyte cell line C20A4 was transfected with either EFEMP1 siRNA (siEFEMP1) or scrambled siRNA (siCtrl). EFEMP1 and MMP-13 protein expression was analyzed by Western blotting. B Chondrocyte-related gene expression, including SOX9, aggrecan (ACAN), MMP13, type X collagen (COL10A1), and type II collagen (COL2A1), was assessed by quantitative PCR in siEFEMP1- and siCtrl-treated C20A4 cells. C Phosphokinase protein arrays were used to profile phosphorylation changes in intracellular proteins extracted from C20A4 chondrocytes. D Spot intensities exhibiting changes greater than ± 25% between groups were quantified using ImageJ software. Red lines indicate baseline levels from the siCtrl group. E Phosphorylation of p70S6K at threonine 421/serine 424 (T421/T424) was further examined by Western blot analysis. Data are presented as mean ± standard error of the mean (SEM). Statistical significance was determined using an independent-samples t-test. * P < 0.05, ** P < 0.01, and *** P < 0.001

Article Snippet: Samples were incubated overnight at 4 °C with primary antibodies against EFEMP1 (1:100, NBP1-77040; NOVUS, USA), SOX9 (1:50, sc-166505; Santa Cruz Biotechnology, USA), MMP-13 (1:100, GTX100665; GeneTex, USA), aggrecan fragments (1:100, AF1220; R&D Systems, USA), COL2A1 (1:100, ab34712; Abcam, UK), and COL10A1 (1:100, GTX37732; GeneTex, USA).

Techniques: Knockdown, Small Interfering RNA, Transfection, Expressing, Western Blot, Gene Expression, Real-time Polymerase Chain Reaction, Phospho-proteomics, Software

Journal: Journal of Molecular Medicine (Berlin, Germany)

Article Title: Targeting EFEMP1 enhances chondrogenesis and inhibits hypertrophic differentiation in a spontaneous osteoarthritis mouse model

doi: 10.1007/s00109-026-02656-y

Figure Lengend Snippet: Immunofluorescence analysis of cartilage markers following EFEMP1 antibody treatment in STR/ort mice. A Representative immunofluorescence (IF) staining for EFEMP1, SOX9, MMP13, aggrecan fragments, COL2A1, and COL10A1 in the medial femoral condyle (MF) and medial tibial plateau (MT). Nuclei were counterstained with DAPI (blue), and articular cartilage regions are outlined by orange dashed lines. B Quantification of fluorescence intensity in articular cartilage regions of the femoral condyle and tibial plateau, with medial and lateral compartments combined. EFEMP1 and SOX9 signals were normalized to DAPI, whereas MMP13, aggrecan fragments, COL10A1, and COL2A1 signals were quantified as absolute FITC intensity. Data are presented as mean ± standard error of the mean (SEM). Statistical analysis was performed using an independent-samples t -test. P < 0.05, P < 0.01, and ** P < 0.001

Article Snippet: Samples were incubated overnight at 4 °C with primary antibodies against EFEMP1 (1:100, NBP1-77040; NOVUS, USA), SOX9 (1:50, sc-166505; Santa Cruz Biotechnology, USA), MMP-13 (1:100, GTX100665; GeneTex, USA), aggrecan fragments (1:100, AF1220; R&D Systems, USA), COL2A1 (1:100, ab34712; Abcam, UK), and COL10A1 (1:100, GTX37732; GeneTex, USA).

Techniques: Immunofluorescence, Staining, Fluorescence

Journal: Pharmaceuticals (Basel, Switzerland)

Article Title: Plastrum testudinis Ameliorates Oxidative Stress in Nucleus Pulposus Cells via Downregulating the TNF-α Signaling Pathway.

doi: 10.3390/ph16101482

Figure Lengend Snippet: Figure 4. Expression of ACAN and COL2A1 in NPCs. (A) Expression of ACAN and COL2A1 pro- teins in NPCs treated with TBHP, with or without PT. (B,C) Expression of ACAN and COL2A1 proteins relative to β-actin in NPCs treated with TBHP, with or without PT. (D) Representa- tive immunofluorescence images of ACAN in NPCs photographed by fluorescence microscopy (scale bar = 100 µm). (E) Representative immunofluorescence images of COL2A1 in NPCs pho- tographed by fluorescence microscopy (scale bar = 100 µm). Data are the mean ± standard deviation. #### p < 0.0001 vs. CTL group; ** p < 0.01, *** p < 0.001, and **** p < 0.0001 vs. 100 µM TBHP group.

Article Snippet: ACAN (sc-166951) and COL2A1 (sc-52658) were from Santa Cruz (Shanghai, China), and prostaglandin G/H synthase 2 (PTGS2) (WL01750), endothelin-1 (EDN1) (WL02780), JUN (WL02863), FOS (WL03699), and IL-6 (WL02841) were from Wanleibio Co. Ltd. (Shenyang, China).

Techniques: Expressing, Microscopy, Standard Deviation

Journal: Pharmaceuticals (Basel, Switzerland)

Article Title: Plastrum testudinis Ameliorates Oxidative Stress in Nucleus Pulposus Cells via Downregulating the TNF-α Signaling Pathway.

doi: 10.3390/ph16101482

Figure Lengend Snippet: Figure 7. Mechanism underlying the therapeutic effect of PT on IDD. PT could downregulate the expression of inflammation-specific targets including TNF-α, FOS, JUN, PTGS2 and EDN1 on the TNF-α signaling pathway as well as cellular senescence marker p16, inflammatory factor IL-6, and upregulate the expressions of COL2A1 and ACAN, thus leading to a suppressive effect on inflammatory response and oxidative stress in NPCs.

Article Snippet: ACAN (sc-166951) and COL2A1 (sc-52658) were from Santa Cruz (Shanghai, China), and prostaglandin G/H synthase 2 (PTGS2) (WL01750), endothelin-1 (EDN1) (WL02780), JUN (WL02863), FOS (WL03699), and IL-6 (WL02841) were from Wanleibio Co. Ltd. (Shenyang, China).

Techniques: Expressing, Marker

Journal: Frontiers in Neuroanatomy

Article Title: Transmitter and ion channel profiles of saccadic omnipause neurons and cholinergic non-omnipause neurons in human nucleus raphe interpositus

doi: 10.3389/fnana.2025.1670220

Figure Lengend Snippet: Immunoperoxidase-based histochemical classification of omnipause neurons (OPN) and cholinergic non-OPNs in human nucleus raphe interpositus (RIP). (A) Combined immunoperoxidase labeling of perineuronal net (PN) marker aggrecan (ACAN, black) and non-phosphorylated neurofilament marker SMI32 (brown) reveals OPNs that are arranged as two columns around the midline in human RIP. The box indicates the area illustrated at higher magnification in B. (B) Close-up demonstrating OPNs co-immunolabeled with ACAN and SMI32 (red arrows) Note the rather dispersed arrangement of OPNs around the midline compared to OPNs in monkey (C) . Also note several neurons that are not ensheathed by PN marker ACAN but express SMI32-immunolabelling (green arrows). (C) In monkey RIP, combined peroxidase labeling of PN marker ACAN (black) and ChAT (brown) demonstrates the absence of such cholinergic neurons and tightly organized OPN columns around the midline. (D) Combined peroxidase labeling of perineuronal net (PN) marker aggrecan (ACAN, black) and non-phosphorylated neurofilament marker SMI32 (brown) and PN marker CSPG (black) choline acetyltransferase (ChAT, brown) reveal the cholinergic non-OPN population (green arrow) that are not ensheathed by PNs. Scale bar represents 1 mm A , 100 μm B,C and 50 μm in D .

Article Snippet: ACAN , Mouse/Monoclonal , Aggrecan , Acris Antibodies GmbH, 32052 Herford, GERMANY , AB_972582 , 1:75.

Techniques: Labeling, Marker, Immunolabeling

Journal: Journal of Tissue Engineering

Article Title: High BMPR2 expression leads to enhanced SMAD1/5/8 signalling and GDF6 responsiveness in human adipose-derived stem cells: implications for stem cell therapies for intervertebral disc degeneration

doi: 10.1177/2041731420919334

Figure Lengend Snippet: Immunohistochemical analysis of ASC collagen gel cultures after 14 days with 100 ng mL −1 rhGDF6 stimulation with and without SMAD1/5/8 (Dorso) or ERK1/2 (U0126) inhibition and no stimulation control. After 14 days culture, constructs stimulated with rhGDF6 demonstrated an increase in aggrecan and type II collagen deposition compared to rhGDF6 stimulated constructs where SMAD1/5/8 and ERK1/2 signalling had been blocked, where levels appeared similar to unstimulated controls. Scale bar, 500 µm.

Article Snippet: Sections were allowed to equilibrate to room temperature, formalin fixed and blocked prior to addition of either aggrecan (Bio-Rad; MCA1454G) or type II collagen (Proteintech 15943-1-AP) primary antibodies and incubated at 4°C overnight.

Techniques: Immunohistochemical staining, Inhibition, Control, Construct

Journal: Stem cell research & therapy

Article Title: Intra-renal delivery of mesenchymal stem cells attenuates myocardial injury after reversal of hypertension in porcine renovascular disease.

doi: 10.1186/scrt541

Figure Lengend Snippet: Figure 1 Hemodynamic effect of renal artery stenosis and characteristics of MSC. A) Renal angiography before and after PTRA, showing successful restoration of vessel patency (arrows). B) Mean arterial pressure (MAP) measured by telemetry decreased after PTRA. C) Immunostaining (left) and FACS (right) showing that adipose tissue-derived MSC expressed CD44 and CD90 surface markers. D) Representative immunostaining showing that MSC trans-differentiated into osteocytes, chondrocytes and adipocytes in vitro. E) DiI-labeled MSC (red) incorporated into tubes formed by HUVEC (grey). F) Representative fluorescence of CM-DiI-labeled MSC (red, arrows) and cytokeratin (green) in the stenotic-kidney (left, X20) and myocardium (right, X40) four weeks after administration. FACS, fluorescence activated cell sorting; HUVEC, human umbilical vein endothelial cells; MSC, mesenchymal stem cells; PTRA, percutaneous transluminal renal angioplasty.

Article Snippet: MSC characterization was confirmed by their trans-differentiation into osteocytes (mouse anti-human osteocalcin antibody and alizarin red staining), chondrocytes (goat anti-human aggrecan antibody) and adipocytes (goat anti-mouse FABP4 antibody and oil red staining) (R&D Systems, Pittsburgh, PA, USA) [17].

Techniques: Immunostaining, Derivative Assay, In Vitro, Labeling, Fluorescence, FACS

Journal: Osteoarthritis and cartilage

Article Title: Development of a novel clinical biomarker assay to detect and quantify aggrecanase-generated aggrecan fragments in human synovial fluid, serum and urine.

doi: 10.1016/j.joca.2010.06.011

Figure Lengend Snippet: Fig. 3. (a) Affinity of the BC3 and BC3-C2 single-chain antibodies for binding to the ARGS neoepitope. The mean fluorescent intensity (MFI) of the antigen-binding channel was measured after incubation with increasing concentrations of ARGS-Bt peptide antigen. Minimal antigen binding is observed with BC3 at the highest antigen concentration (3.2 mM). (b) Affinity of the BC3-C2 single-chain antibody for binding to the rat (ARGN) and human (ARGS) neoepitopes. Quantitative equilibrium binding was determined using flow cytometry. The Ag-binding MFI was normalized and fit to a nonlinear least-squares regression to calculate the equilibrium affinity constant. Bt: biotinylated peptide. (c) Biacore data comparing the affinities of the BC3 and BC3-C2 IgG antibodies. BC3 and BC3-C2 IgG were immobilized and antigen-binding responses measured by surface plasmon resonance using Biacore instrumentation. Representative data from separate experiments were combined to highlight the change in dissociation rate of BC3-C2 after antibody optimization. (d) Western blot analysis of human aggrecan digested with or without ADAMTS-4 and detected with the BC3 or BC3-C2 antibody.

Article Snippet: The amount of aggrecan fragments present in the sample was determined based on a standard curve generated with ADAMTS-4 digested recombinant G1-IGD-G2 of aggrecan (that gets cleaved into G1—NITEGE and ARGS—G2 fragments by ADAMTS-4) (catalog number 1220-PG-025, R&D Systems, Minneapolis, MN).

Techniques: Binding Assay, Incubation, Concentration Assay, Cytometry, SPR Assay, Western Blot

Journal: Osteoarthritis and cartilage

Article Title: Development of a novel clinical biomarker assay to detect and quantify aggrecanase-generated aggrecan fragments in human synovial fluid, serum and urine.

doi: 10.1016/j.joca.2010.06.011

Figure Lengend Snippet: Fig. 4. Comparison of the BC3 and BC3-C2 antibodies for capturing human aggrecan in the sandwich ELISA. Varying amounts of native human aggrecan ADAMTS-4 digestion were analyzed by sandwich ELISA using the BC3 or BC3-C2 antibody as capture antibody. NHA: native human aggrecan; TS-4: ADAMTS-4/aggrecanase-1.

Article Snippet: The amount of aggrecan fragments present in the sample was determined based on a standard curve generated with ADAMTS-4 digested recombinant G1-IGD-G2 of aggrecan (that gets cleaved into G1—NITEGE and ARGS—G2 fragments by ADAMTS-4) (catalog number 1220-PG-025, R&D Systems, Minneapolis, MN).

Techniques: Comparison, Sandwich ELISA