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: Journal of Orthopaedic Translation

Article Title: USP39 inhibits MLKL phosphorylation and deubiquitination to suppress necroptosis of nucleus pulposus cells and attenuate intervertebral disc degeneration

doi: 10.1016/j.jot.2025.101039

Figure Lengend Snippet: Knockdown of MLKL alleviated IVDD in TSZ stimulation model of NPCs. (A) Western blot analysis showing MLKL knockdown efficiency using sh1-MLKL, sh2-MLKL, and sh3-MLKL in NPCs (n = 3 per group). (B, C) Representative fluorescence images and quantification of MLKL expression in Control and sh-MLKL groups (n = 3 per group; scale bar = 50 μm). (D) PCR analysis of ACAN and MMP3 expression in NPCs from Control, TSZ, and TSZ + sh-MLKL groups (n = 3 per group). (E, F) Western blot analysis of ACAN and MMP3 protein levels in Control, TSZ, and TSZ + sh-MLKL groups (n = 3 per group). (G, H) Representative fluorescence images and quantification of ACAN and MMP3 expression in Control, TSZ, and TSZ + sh-MLKL groups (n = 3 per group; scale bar = 50 μm).∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Article Snippet: Resolved proteins were transferred to 0.45-μm polyvinylidene difluoride membranes (IPVH00010, Millipore, USA) via wet transfer at 300 mA for 1.5–2 h. Membranes were blocked with protein-free rapid blocking buffer (PS108P, Epizyme, China) for 1 h at room temperature, followed by incubation at 4 °C overnight with primary antibodies targeting: USP39 (A2389, Abclonal, China,1:500), ACAN (DF7561, Affinity, China, 1:500), MMP3 (A11418, Abclonal, China, 1:1000), MLKL (A21894, Abclonal, China, 1:1000), p-MLKL (AP1244, Abclonal, China, 1:200), Ubiquitin (A19686,Abclonal, China, 1:1000), GAPDH (AC002, Abclonal, China, 1:15000).

Techniques: Knockdown, Western Blot, Fluorescence, Expressing, Control

Journal: Journal of Orthopaedic Translation

Article Title: USP39 inhibits MLKL phosphorylation and deubiquitination to suppress necroptosis of nucleus pulposus cells and attenuate intervertebral disc degeneration

doi: 10.1016/j.jot.2025.101039

Figure Lengend Snippet: Conditional knockout of Mlkl inhibited the process of IVDD in mice. A) Schematic diagram of Mlkl CKO mouse generation. (B, C) Schematic illustrations of the LSI surgery model and the aging mouse model. (D, E) Representative IVD sections stained with H&E and SF from WT and Mlkl CKO groups after 8 weeks of LSI exposure (n = 5 per group; scale bars = 200 μm and 50 μm). (F, G) Representative IVD sections stained with H&E and SF from WT and Mlkl CKO groups in the aging model (n = 5 per group; scale bars = 200 μm and 50 μm). (H) Representative fluorescence images and quantification of Acan and Mmp3 expression in WT and Mlkl CKO groups following LSI (n = 5 per group; scale bars = 200 μm and 50 μm). (I) Representative fluorescence images and quantification of Acan and Mmp3 expression in WT and Mlkl CKO groups in the aging model (n = 5 per group; scale bars = 200 μm and 50 μm).

Article Snippet: Resolved proteins were transferred to 0.45-μm polyvinylidene difluoride membranes (IPVH00010, Millipore, USA) via wet transfer at 300 mA for 1.5–2 h. Membranes were blocked with protein-free rapid blocking buffer (PS108P, Epizyme, China) for 1 h at room temperature, followed by incubation at 4 °C overnight with primary antibodies targeting: USP39 (A2389, Abclonal, China,1:500), ACAN (DF7561, Affinity, China, 1:500), MMP3 (A11418, Abclonal, China, 1:1000), MLKL (A21894, Abclonal, China, 1:1000), p-MLKL (AP1244, Abclonal, China, 1:200), Ubiquitin (A19686,Abclonal, China, 1:1000), GAPDH (AC002, Abclonal, China, 1:15000).

Techniques: Knock-Out, Staining, Fluorescence, Expressing

Journal: Journal of Orthopaedic Translation

Article Title: USP39 inhibits MLKL phosphorylation and deubiquitination to suppress necroptosis of nucleus pulposus cells and attenuate intervertebral disc degeneration

doi: 10.1016/j.jot.2025.101039

Figure Lengend Snippet: USP39 alleviated IVDD by inhibiting ubiquitination and phosphorylation of MLKL. (A, B) Western blot analysis of USP39 knockdown efficiency (n = 3 per group). (C, D) Western blot analysis of USP39 overexpression efficiency (n = 3 per group). (E) Western blot analysis of MLKL ubiquitination and phosphorylation levels in Control, TSZ, TSZ + sh-USP39, and TSZ + oe-USP39 groups (n = 3 per group). (F) PCR analysis of ACAN and MMP3 expression in NPCs from Control, TSZ, TSZ + sh-USP39, and TSZ + oe-USP39 groups (n = 3 per group). (G, H) Western blot analysis of USP39, ACAN, and MMP3 expression in Control, TSZ, TSZ + sh-USP39, and TSZ + oe-USP39 groups (n = 3 per group). (I) Representative immunofluorescence staining and fluorescence intensities of ACAN and MMP3 in NPCs from Control, TSZ, TSZ + sh-USP39, and TSZ + oe-USP39 groups (n = 3 per group; scale bar = 50 μm). (J) Expression levels of ACAN and MMP3 in Control, TSZ, TSZ + sh-USP39, TSZ + sh-USP39+sh-MLKL, and TSZ + sh-USP39+NSA groups (n = 3 per group). (K) Representative immunofluorescence staining and fluorescence intensities of ACAN and MMP3 in NPCs from Control, TSZ, TSZ + sh-USP39, TSZ + sh-USP39+sh-MLKL, and TSZ + sh-USP39+NSA groups (n = 3 per group; scale bar = 50 μm). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Article Snippet: Resolved proteins were transferred to 0.45-μm polyvinylidene difluoride membranes (IPVH00010, Millipore, USA) via wet transfer at 300 mA for 1.5–2 h. Membranes were blocked with protein-free rapid blocking buffer (PS108P, Epizyme, China) for 1 h at room temperature, followed by incubation at 4 °C overnight with primary antibodies targeting: USP39 (A2389, Abclonal, China,1:500), ACAN (DF7561, Affinity, China, 1:500), MMP3 (A11418, Abclonal, China, 1:1000), MLKL (A21894, Abclonal, China, 1:1000), p-MLKL (AP1244, Abclonal, China, 1:200), Ubiquitin (A19686,Abclonal, China, 1:1000), GAPDH (AC002, Abclonal, China, 1:15000).

Techniques: Ubiquitin Proteomics, Phospho-proteomics, Western Blot, Knockdown, Over Expression, Control, Expressing, Immunofluorescence, Staining, Fluorescence

Journal: Journal of Orthopaedic Translation

Article Title: USP39 inhibits MLKL phosphorylation and deubiquitination to suppress necroptosis of nucleus pulposus cells and attenuate intervertebral disc degeneration

doi: 10.1016/j.jot.2025.101039

Figure Lengend Snippet: Overexpression of Usp39 attenuated in vivo IVDD progression by suppressing MLKL phosphorylation. (A) Schematic illustration of AAV-Usp39 injection following LSI. (B) RT-qPCR analysis confirming significantly increased Usp39 mRNA levels in NP tissues following AAV-Usp39 injection. (C) Representative lumbar spine MRI of mice at 8 weeks in Sham (n = 5), LSI (n = 5), and LSI + AAV-Usp39 (n = 5) groups. (D, E) Representative H&E and SF staining of spinal tissues from Sham, LSI, and LSI + AAV-Usp39 groups (n = 5 per group; scale bars = 200 μm and 50 μm). (F–H) Representative immunofluorescence staining of Acan, Mmp3, and p-Mlkl in Sham, LSI, and LSI + AAV-Usp39 groups (n = 5 per group; scale bars = 200 μm and 50 μm).

Article Snippet: Resolved proteins were transferred to 0.45-μm polyvinylidene difluoride membranes (IPVH00010, Millipore, USA) via wet transfer at 300 mA for 1.5–2 h. Membranes were blocked with protein-free rapid blocking buffer (PS108P, Epizyme, China) for 1 h at room temperature, followed by incubation at 4 °C overnight with primary antibodies targeting: USP39 (A2389, Abclonal, China,1:500), ACAN (DF7561, Affinity, China, 1:500), MMP3 (A11418, Abclonal, China, 1:1000), MLKL (A21894, Abclonal, China, 1:1000), p-MLKL (AP1244, Abclonal, China, 1:200), Ubiquitin (A19686,Abclonal, China, 1:1000), GAPDH (AC002, Abclonal, China, 1:15000).

Techniques: Over Expression, In Vivo, Phospho-proteomics, Injection, Quantitative RT-PCR, Staining, Immunofluorescence

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: Nutrients

Article Title: CHL Attenuates MIA-Induced Osteoarthritis by Regulating Inflammatory, Catabolic, and Anabolic Pathways Associated with Cartilage and Subchondral Bone Degeneration

doi: 10.3390/nu18183031

Figure Lengend Snippet: Effects of CHL on serum biomarkers associated with cartilage degradation and ECM metabolism. SD rats were randomly divided into six groups ( n = 8/group): NC, MIA, MIA + celecoxib (PC, 10 mg/kg/day), and CHL-treated LOW (10 mg/kg/day), MID (30 mg/kg/day), and HIGH (300 mg/kg/day) groups. CHL and celecoxib were administered orally for 6 weeks, with OA induced after 2 weeks of pre-administration by intra-articular injection of MIA. Serum levels of ( A ) COMP, ( B ) CTX-II, ( C ) GAG and ( D ) aggrecan were measured using ELISA kits. Values are presented as the mean ± SD ( n = 8). Statistical significance is indicated by distinct letters, with different letters indicating statistically significant differences between groups. COMP, cartilage oligomeric matrix protein; CTX-II, C-terminal crosslinked telopeptide of type II collagen; and GAG, glycosaminoglycans.

Article Snippet: ELISA kits for GAGs and aggrecan were obtained from Biomatik (Kitchener, ON, Canada).

Techniques: Injection, Enzyme-linked Immunosorbent Assay

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