Journal: The FASEB Journal

Article Title: Amelogenin Null Mice Develop Osteoarthritis, While Its Application Mitigates Disease Phenotypes in a Rat Model

doi: 10.1096/fj.202500827R

Figure Lengend Snippet: Immunohistochemical analyses of the knee joints from 12‐month‐old amelogenin‐null and wild‐type mice. Representative images of immunohistochemistry for type II collagen (A, C) and matrix metalloproteinase‐13 (MMP‐13) (B, D) in wild‐type (WT) (A, B) and amelogenin‐null (C, D) mice. Amelogenin‐null mice exhibited reduced type II collagen levels and increased MMP‐13 expression compared to wild‐type mice. These changes were accompanied by cartilage destruction and the presence of hypertrophic chondrocytes (black arrowheads). Enlarged pictures were taken from the regions marked with black frames. Brown staining indicates positive expression of type II collagen or MMP‐13. Scale bars: 250 μm for overall images; 62.5 μm for enlarged images.

Article Snippet: Indirect immunohistochemistry was performed using primary antibodies directed against type II collagen (AM26374PU‐N, ORIGENE) or matrix metalloproteinase 13 (MMP‐13; TA321485, ORIGENE).

Techniques: Immunohistochemical staining, Immunohistochemistry, Expressing, Staining

Journal: The journal of gene medicine

Article Title: Sp2 Transcription Factor Alleviates Chondrocyte Loss in Osteoarthritis by Repressing the DVL1-Dependent Wnt/β-Catenin Signaling Pathway.

doi: 10.1002/jgm.70021

Figure Lengend Snippet: FIGURE 4 | CHIR-99021 restores cartilage injury mitigated by DVL1 silencing. Mice stably administered sh-DVL1 were further treated with the Wnt/β-catenin agonist CHIR-99021 via intra-articular injection. (A) Protein levels of β-catenin in cells determined using WB analysis. (B) Cartilage morphology in the mouse knee joints determined using Safranin O/fast green staining. (C) Protein levels of COL10A1 and COL2A1 in mouse chon- drocytes determined using WB analysis. (D) Positive TRAP staining in the mouse knee joints; in vitro, chondrocytes with stable DVL1 knockdown were treated with 5 μM CHIR-99021 for 24 h. (E) Apoptosis in the extracted chondrocytes determined using TUNEL assay. (F) Protein levels of pro- cleaved-caspase-3 in the extracted chondrocytes determined using WB analysis. (G) Protein levels of expression of MMP13 and SOX9 in the mouse chondrocytes determined using WB analysis. For animal experiments, each group contained five mice. For cell experiments, three biological repli- cates were performed. Differences were compared by the unpaired t-test (A–G).

Article Snippet: The membranes were blocked at room temperature for 1 h in Tris- buffered saline (T5912, Merck) and incubated with antibodies against DVL1 (1:1000, 13- 706, ProSci), COL10A1 (collagen type X alpha 1 chain) (1:1000, A11645, ABclonal Technology Co. Ltd., Wuhan, Hubei, China), MMP13 (matrix metallopeptidase 13) (1:4000, ab39012, Abcam), SOX9 (SRY- box transcription factor 9) (1:1000, orb1258160, Biorbyt LLC, San Francisco, California, United States), cleavedcaspase- 3 (1:1000, PA5- 114687, Thermo Fisher Scientific), βcatenin (1:5000, ab32572, Abcam), COL2A1 (collagen type II alpha 1 chain) (1:1000, orb1259434, Biorbyt), SP2 (1:1000, PA5- 103254, Thermo Fisher Scientific), and GAPDH (1:2500, ab9485, Abcam) overnight at 4°C.

Techniques: Stable Transfection, Injection, Staining, In Vitro, Knockdown, TUNEL Assay, Expressing

Journal: The journal of gene medicine

Article Title: Sp2 Transcription Factor Alleviates Chondrocyte Loss in Osteoarthritis by Repressing the DVL1-Dependent Wnt/β-Catenin Signaling Pathway.

doi: 10.1002/jgm.70021

Figure Lengend Snippet: FIGURE 7 | DVL1 overexpression increases chondrocyte loss suppressed by SP2. Chondrocytes were administered lentiviral vectors encapsu- lating OE-NC/OE-SP2 or the additional OE-NC/OE-DVL1. (A) mRNA expression of DVL1 in cells determined using RT-qPCR. (B) Apoptosis in cells determined using TUNEL assay. (C) Protein levels of β-catenin in chondrocytes determined using WB analysis. (D) Transcriptional activity of β-catenin in chondrocytes analyzed by TOP/FOPFlash assays. (E) Protein levels of SOX9 and COL2A1 in the chondrocytes determined using WB analysis. Three biological replicates were performed. Differences were compared by unpaired t-test (A) or ANOVA (B–E).

Article Snippet: The membranes were blocked at room temperature for 1 h in Tris- buffered saline (T5912, Merck) and incubated with antibodies against DVL1 (1:1000, 13- 706, ProSci), COL10A1 (collagen type X alpha 1 chain) (1:1000, A11645, ABclonal Technology Co. Ltd., Wuhan, Hubei, China), MMP13 (matrix metallopeptidase 13) (1:4000, ab39012, Abcam), SOX9 (SRY- box transcription factor 9) (1:1000, orb1258160, Biorbyt LLC, San Francisco, California, United States), cleavedcaspase- 3 (1:1000, PA5- 114687, Thermo Fisher Scientific), βcatenin (1:5000, ab32572, Abcam), COL2A1 (collagen type II alpha 1 chain) (1:1000, orb1259434, Biorbyt), SP2 (1:1000, PA5- 103254, Thermo Fisher Scientific), and GAPDH (1:2500, ab9485, Abcam) overnight at 4°C.

Techniques: Over Expression, Expressing, Quantitative RT-PCR, TUNEL Assay, Activity Assay

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: Icariin Reduces Cartilage Degeneration in a Mouse Model of Osteoarthritis and is Associated with the Changes in Expression of Indian Hedgehog and Parathyroid Hormone-Related Protein

doi: 10.12659/MSM.910983

Figure Lengend Snippet: Icariin treatment of chondrocytes increased proteoglycan synthesis during chondrogenic differentiation. ( A ) Micromass culture of chondrocytes in the chondrogenic medium without or with different concentration of icariin: 10 −7 M, 10 −6 M, and 10 −5 M. Alcian blue staining was used to detect the proteoglycans synthesis in day 7 and day 14 cells. ( B ) Quantification of the Alcian blue staining in ( A ). ( C ) Enzyme-linked immunosorbent assay (ELISA) results of the expression of collagen type II alpha 1 (COL2A1) of cells in culture on Day 7 and Day 14 treated with or without icariin. Data are presented as the mean ±SD. N=4. NS – nonsignificant * P<0.05; ** P<0.01.

Article Snippet: The levels of Col2a1 protein in cell culture supernatants was measured by using a Col2a1 enzyme-linked immunosorbent assay (ELISA) kit (CSB-EL005739MO) (Cusabio, China), according to the manufacturer’s instructions.

Techniques: Concentration Assay, Staining, Enzyme-linked Immunosorbent Assay, Expressing