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: Journal of Biological Chemistry

Article Title: Cyclosporin A Prevents the Hypoxic Adaptation by Activating Hypoxia-inducible Factor-1α Pro-564 Hydroxylation

doi: 10.1074/jbc.m211293200

Figure Lengend Snippet: FIG. 5. CsA activates Pro-564 hydroxylase. A kidney light mito- chondrial fraction was used as a source of enzyme. Activity was defined as the Pro-564 peptide-dependent degradation of 2-OG. The peptide- independent activity was determined in each experiment and was sub- strated from the data. Means S.E. from triplicate measurements are shown. A, 2-OG dependence. Experiments were performed in the pres- ence of 100 M Pro-564 peptide. B, dependence on Pro-564 peptide. Experiments were performed in the absence (G) or the presence (Œ) of 10 M CsA. C, dose-response curve for the stimulating action of CsA. Experiments were performed in the presence of 50 M Pro-564 peptide. D, vHL binding. GSTODD (170 g) was incubated for 30 min at 37 °C in the presence of the kidney extract and cofactors. Glutathione-Sepha- rose beads and 50,000 cpm of [35S]vHL were then added. The bound vHL was recovered after 2 h at 4 °C, subjected to 15% SDS-PAGE, and visualized by phosphorimaging. The left lane shows a band from di- rectly loaded [35S]vHL. E, in vivo hydroxylation of ODDGFP. ODDGFP-expressing cells were incubated under normoxia or hypoxia in the presence or absence of 10 M CsA. ODDGFP was immunopre- cipitated by using an anti-GFP monoclonal antibody, resolved by SDS- PAGE, and probed with an anti-vHL (upper panel) or an anti-GFP (lower panel) monoclonal antibodies. Note than in this experiment, the expression of ODDGFP was stabilized under hypoxia and reversed by addition of CsA. Results shown are representative of two other inde- pendent experiments.

Article Snippet: Mouse monoclonal antibodies were obtained from the following sources: HIF-1 (Novus Biologicals), GFP (Roche Molecular Biochemicals), -tubulin (Sigma), vHL (Pharmingen).

Techniques: Activity Assay, Binding Assay, Incubation, SDS Page, In Vivo, Expressing, Bioprocessing