aggrecan fragmentation  (Thermo Fisher)

Journal: International Journal of Molecular Sciences

Article Title: Ionizing Radiation Induces Disc Annulus Fibrosus Senescence and Matrix Catabolism via MMP-Mediated Pathways

doi: 10.3390/ijms23074014

Figure Lengend Snippet: Senescence and matrix breakdown in Human AF Cells. Depicts results from in vitro human AF cell ionizing radiation experimentation. ( A ) Morphology depicts changes in cellular appearance after the administration of 15 Gy ionizing radiation visible on light microscopy. ( B ) Senescence-associated β-galactosidase assay with culture morphology in light microscopy (left) and proportion of assay-positive cells indicating senescence with 15 Gy ionizing radiation dose. ( C ) WB for Aggrecan Fragmentation depicts 3 control and 3 ionizing radiation western blot results for aggrecan fragmentation mediated by ADAMTS (75 kDA) or MMP (55 kDA) (left). Quantification of band intensities from western blot ADAMTS and MMP fragmentation are depicted (right) with 15 Gy dose of ionizing radiation and 0 Gy dose control. On microscopic images, the white bar for scale measures 20 μm. * p ≤ 0.05.

Article Snippet: Disc protein extracts were then used for western blot for detection of aggrecan fragmentation, using Tris-HEPES 4–20% gradient gel (Thermo Scientific 25204, Waltham, MA, USA), Tris-HEPES-SDS Running Buffer (Thermo 28398), Tris-Glycine Transfer Buffer with 10% Methanol (Thermo Scientific 28380, Fisher Scientific A452-4), and TBST (Sigma-Aldrich T9039).

Techniques: In Vitro, Light Microscopy, Control, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Ionizing Radiation Induces Disc Annulus Fibrosus Senescence and Matrix Catabolism via MMP-Mediated Pathways

doi: 10.3390/ijms23074014

Figure Lengend Snippet: IR treatment increased matrix breakdown in rat AF cells. Rat AF cell cultures were treated with 15 Gy IR and incubated for 5 days to establish senescence. ( A ) Western blot (WB) analysis of anti-aggrecan fragmentation from 3 control and 3 IR AF samples showed elevated anti-aggrecan fragmentation mediated by ADAMTS (75 kDA) and MMP (55 kDa) in IR-treated AF cell samples. “C” designates controls, “IR” designates ionizing radiation treated, and “L” designates protein ladder. Left side, a schematic representation of aggregate consisting of the core aggrecan protein bound to GAG side chains at the chondroitin sites (CS-1, CS-2), and the MMP- and ADAMTS-mediated cleavage site within the interglobular domain residing between the G1 and G2 domain of aggrecan are indicated with arrows. Right side, quantification of western blot band intensity is depicted in graphs on right. ( B ) IF staining demonstrated elevated MMP1 and MMP3 protein expression (red) in IR-treated AF cells compared to untreated cells. ( C ) qRT-PCR also confirmed elevated MMP-1 and -3 gene expression in IR-treated AF cells compared to untreated cells. Data shown as an average of n = 3 with one standard deviation. On microscopic images, the white bar for scale measures 20 μm. * p ≤ 0.05.

Article Snippet: Disc protein extracts were then used for western blot for detection of aggrecan fragmentation, using Tris-HEPES 4–20% gradient gel (Thermo Scientific 25204, Waltham, MA, USA), Tris-HEPES-SDS Running Buffer (Thermo 28398), Tris-Glycine Transfer Buffer with 10% Methanol (Thermo Scientific 28380, Fisher Scientific A452-4), and TBST (Sigma-Aldrich T9039).

Techniques: Incubation, Western Blot, Control, Staining, Expressing, Quantitative RT-PCR, Gene Expression, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: Ionizing Radiation Induces Disc Annulus Fibrosus Senescence and Matrix Catabolism via MMP-Mediated Pathways

doi: 10.3390/ijms23074014

Figure Lengend Snippet: IR treatment effects on matrix anabolism in rat AF cells. Rat AF cell cultures were treated with 15 Gy IR and incubated for 5 days to establish senescence. ( A ) qRT-PCR revealed increased mRNA expression of collagen-1, collagen-2, and aggrecan in IR-treated AF cells compared to untreated AF cells gene expression. ( B ) Immunofluorescence showed increased collagen-2 but unchanged collagen-1 and aggrecan protein expression with 15 Gy of ionizing radiation administration. ( C ) DMMB assay for total glycosaminoglycan (GAG) content showed a decrease in GAG in IR-treated AF cells compared to untreated control. Data shown as an average of n = 3 with one standard deviation. * p ≤ 0.05.

Article Snippet: Disc protein extracts were then used for western blot for detection of aggrecan fragmentation, using Tris-HEPES 4–20% gradient gel (Thermo Scientific 25204, Waltham, MA, USA), Tris-HEPES-SDS Running Buffer (Thermo 28398), Tris-Glycine Transfer Buffer with 10% Methanol (Thermo Scientific 28380, Fisher Scientific A452-4), and TBST (Sigma-Aldrich T9039).

Techniques: Incubation, Quantitative RT-PCR, Expressing, Gene Expression, Immunofluorescence, Dimethylmethylene Blue Assay, Control, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: Ionizing Radiation Induces Disc Annulus Fibrosus Senescence and Matrix Catabolism via MMP-Mediated Pathways

doi: 10.3390/ijms23074014

Figure Lengend Snippet: IR treatment reduced aggrecan gene expression in mouse intervertebral discs. RT-PCR quantification of murine aggrecan relative gene expression after the administration of 3 Gy ionizing radiation is depicted compared to control. Data shown as an average of n = 5 with one standard deviation. * p ≤ 0.05.

Article Snippet: Disc protein extracts were then used for western blot for detection of aggrecan fragmentation, using Tris-HEPES 4–20% gradient gel (Thermo Scientific 25204, Waltham, MA, USA), Tris-HEPES-SDS Running Buffer (Thermo 28398), Tris-Glycine Transfer Buffer with 10% Methanol (Thermo Scientific 28380, Fisher Scientific A452-4), and TBST (Sigma-Aldrich T9039).

Techniques: Gene Expression, Reverse Transcription Polymerase Chain Reaction, Control, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: Ionizing Radiation Induces Disc Annulus Fibrosus Senescence and Matrix Catabolism via MMP-Mediated Pathways

doi: 10.3390/ijms23074014

Figure Lengend Snippet: IR treatment increased disc aggrecanolysis in mice. A representative western blot of whole- disc tissue extract showing aggrecan fragments generated from MMP-mediated (55 kDa band) and ADAMTS-mediated (75 kDa band) activities are shown (left) with quantitative band intensity results (right) exhibited. Western blot “L” designates protein ladder with “0 Gy” and “3 Gy” representing radiation treatment dose groups. Data shown as an average of n = 5 with one standard deviation. * p ≤ 0.05.

Article Snippet: Disc protein extracts were then used for western blot for detection of aggrecan fragmentation, using Tris-HEPES 4–20% gradient gel (Thermo Scientific 25204, Waltham, MA, USA), Tris-HEPES-SDS Running Buffer (Thermo 28398), Tris-Glycine Transfer Buffer with 10% Methanol (Thermo Scientific 28380, Fisher Scientific A452-4), and TBST (Sigma-Aldrich T9039).

Techniques: Western Blot, Generated, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: Ionizing Radiation Induces Disc Annulus Fibrosus Senescence and Matrix Catabolism via MMP-Mediated Pathways

doi: 10.3390/ijms23074014

Figure Lengend Snippet: qRT-PCR primers used for quantifying genes involved in senescence and matrix anabolism and catabolism. Forward (FW) and reverse (RV) sequences for human PCR primer genes used in qRT-PCR assays for matrix proteins, matrix catabolic proteins, and cellular senescence markers. A, T, C, and G letters in sequences represent base pairs.

Article Snippet: Disc protein extracts were then used for western blot for detection of aggrecan fragmentation, using Tris-HEPES 4–20% gradient gel (Thermo Scientific 25204, Waltham, MA, USA), Tris-HEPES-SDS Running Buffer (Thermo 28398), Tris-Glycine Transfer Buffer with 10% Methanol (Thermo Scientific 28380, Fisher Scientific A452-4), and TBST (Sigma-Aldrich T9039).

Techniques: Sequencing

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