fibulin 3  (R&D Systems)

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: Effect of EFEMP1 knockdown on osteoblast differentiation and mineralization in preosteoblastic cells. A The preosteoblastic MC3T3-E1 cell line was transfected with either EFEMP1 siRNA (siEFEMP1) or scrambled siRNA (siCtrl). EFEMP1 and RUNX2 protein expression was analyzed by Western blotting. B, C Alizarin Red staining was used to assess matrix mineralization in MC3T3-E1 cells cultured in osteogenic induction medium (OIM), with quantification performed on days 21, 28, and 35. D Alkaline phosphatase (ALP) staining was performed to evaluate osteogenic differentiation. E ALP enzymatic activity was measured in cell lysates on days 9 and 14 following siRNA transfection. F Osteopontin (OPN) expression was analyzed on days 3 and 7 during osteogenic induction. Data are presented as mean ± standard error of the mean (SEM). Statistical analyses: Data in A were analyzed using an independent-samples t -test, while data in C , E , and F were analyzed using one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison 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, Transfection, Expressing, Western Blot, Staining, Cell Culture, Activity Assay, Comparison

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: EFEMP1 antibody treatment and cartilage histological assessment in a mouse model of osteoarthritis (OA). A Schematic illustration of the EFEMP1 antibody treatment protocol in STR/ort mice from 16 to 21 weeks of age. B Representative Safranin O–stained knee joint sections, with magnified views of the non-calcified cartilage (NCC) and calcified cartilage (CC) regions. Matrix-producing chondrocytes (MPCs) and matrix-non-producing chondrocytes (MNCs) are indicated by arrows. C Quantification of MPC and MNC numbers in the NCC and CC regions of articular cartilage, with counts combined from medial and lateral compartments. D Osteoarthritis severity was evaluated using Osteoarthritis Research Society International (OARSI) scores across four joint compartments: lateral femoral condyle (LF), medial femoral condyle (MF), lateral tibial plateau (LT), and medial tibial plateau (MT). Data in C are presented as mean ± standard error of the mean (SEM), while data in D are presented as median and quartiles using a violin plot. Statistical analysis was performed using an independent-samples t-test. * P < 0.05 and ** P < 0.01

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: Staining

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 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: Cytokine profiling following EFEMP1 antibody treatment in STR/ort mice with osteoarthritis. A Heat-map representation of serum cytokine expression profiles measured using a multiplex assay. B Quantification of serum cytokine concentrations determined by Luminex xMAP analysis. Data are presented as mean ± standard error of the mean (SEM). Statistical analysis was performed using one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparisons 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: Expressing, Multiplex Assay, Luminex

Journal: JHEP Reports

Article Title: Biliary atresia susceptibility gene EFEMP1 regulates extrahepatic bile duct elastic fiber formation and mechanics

doi: 10.1016/j.jhepr.2024.101215

Figure Lengend Snippet: Elastic fibers are reduced in adult Efemp1 +/- EHBDs but elastin expression is unchanged. (A) Representative staining of elastin in WT and Efemp1 +/- EHBDs. (B) Quantification of percent area of staining. (C) Representative elastic fiber staining of WT and Efemp1 +/- EHBDs. Arrowheads show examples of elastic fibers. Note organized fibers under the basement membrane in WT. Additional examples shown in Fig. S2 . (D) Quantification of the number of elastic fibers per 50 μm 2 . (E) Representative images of collagen by SHG imaging in WT and Efemp1 +/- EHBDs. (F) Quantification of percent collagen area of SHG signal. Forward signal in red and backward signal in green. Dotted lines and L denote lumens. 6-8 ducts were analyzed per genotype for all stains, with 3-5 images taken per duct. All scale bars 50 μm. Data shown are mean ± SEM. Significance in B and D determined by unpaired t-test. Significance in F determined using the Mann-Whitney test since the data were not normally distributed. EHBD, extrahepatic bile duct; SHG, second harmonic generation; WT, wild-type.

Article Snippet: Heat-mediated antigen retrieval with 10 mM citric acid pH 6.0 in a pressure cooker for 2 h was used for staining with antibodies against EFEMP1 (fibulin-3) (1:100, Thermo Fisher, Waltham, MA, #PA5-29347), fibulin-4 (1:100, Abcam, Waltham, MA, #Ab1250730), or fibulin-5 (1:200, Proteintech, Rosement, IL, #12188-1-AP).

Techniques: Expressing, Staining, Membrane, Imaging, MANN-WHITNEY

Journal: JHEP Reports

Article Title: Biliary atresia susceptibility gene EFEMP1 regulates extrahepatic bile duct elastic fiber formation and mechanics

doi: 10.1016/j.jhepr.2024.101215

Figure Lengend Snippet: Mechanical properties measured by pressure myography are altered in Efemp1 +/- EHBDs. (A) Measured outer diameter of WT and Efemp1 +/- adult mice EHBDs at increasing pressures. (B) Calculated circumferential stress of WT and Efemp1 +/- adult mice EHBDs at increasing pressures. (C) Calculated circumferential stretch of WT and Efemp1 +/- adult mice EHBDs at increasing pressures. (D) Relative change in inner diameter of WT vs. Efemp1 +/- treated EHBD as pressure is increased from unloaded (0 mm Hg) condition to physiological pressure (2 mmHg). Data shown are mean ± SEM. Six mice used per condition. ## represent uneven x axes. p values in graphs A-C represent two-way ANOVA between profile of WT vs. Efemp1 +/- . p value in D represents Fisher's least significant difference after two-way ANOVA. EHBD, extrahepatic bile duct; WT, wild-type.

Article Snippet: Heat-mediated antigen retrieval with 10 mM citric acid pH 6.0 in a pressure cooker for 2 h was used for staining with antibodies against EFEMP1 (fibulin-3) (1:100, Thermo Fisher, Waltham, MA, #PA5-29347), fibulin-4 (1:100, Abcam, Waltham, MA, #Ab1250730), or fibulin-5 (1:200, Proteintech, Rosement, IL, #12188-1-AP).

Techniques: