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 2 | DVL1 knockdown reduces β-catenin levels in the cartilage and chondrocytes. Mice with OA were administered adenoviral vectors containing either sh-DVL1 or sh-NC via intra-articular injections. Lentiviral vectors carrying sh-DVL1 or sh-NC were introduced into chondrocytes extracted from OA mice. The mRNA (A) and protein (B) levels of DVL1 in chondrocytes were determined using RT-qPCR and WB analysis, respec- tively. (C) Protein levels of β-catenin in chondrocytes determined using WB analysis. (D) The transcriptional activity of β-catenin in chondrocytes analyzed by TOP/FOPFlash assays. (E) Positive staining of DVL1 in the mouse knee joint determined using immunofluorescence staining. (F) mRNA expression of DVL1 in the joint cartilage determined using RT-qPCR. (G) Positive expression of β-catenin in the joint cartilage determined us- ing IHC assay. For animal experiments, each group contained five mice. For cell experiments, three biological replicates were performed. Differences were compared by the unpaired t-test (C, D) or ANOVA (A, B, F, and G).

Article Snippet: The sections were blocked with 5% goat serum for 1 h, followed by an overnight incubation at 4°C with the antibodies against DVL1 (1:100, 13–706, ProSci, Poway, California, United States), β- catenin (1:500, ab32572, Abcam), and SP2 (1:100, PA5- 103254, Thermo Fisher Scientific).

Techniques: Knockdown, Quantitative RT-PCR, Activity Assay, Staining, Immunofluorescence, 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 3 | DVL1 knockdown ameliorates cartilage injury in mice. (A) Protein levels of COL10A1, MMP13, and SOX9 in mouse cartilage tissues determined using WB analysis. (B) Cartilage morphology in the mouse knee joints determined using Safranin O/fast green staining. (C) Positive TRAP staining in the mouse knee joints. (D) Apoptosis in the extracted chondrocytes determined using TUNEL assay. (E) Protein levels of pro- cleaved-caspase-3 in the extracted chondrocytes determined using WB analysis. (F) Protein levels of COL10A1, 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 (D–F) or ANOVA (A–C).

Article Snippet: The sections were blocked with 5% goat serum for 1 h, followed by an overnight incubation at 4°C with the antibodies against DVL1 (1:100, 13–706, ProSci, Poway, California, United States), β- catenin (1:500, ab32572, Abcam), and SP2 (1:100, PA5- 103254, Thermo Fisher Scientific).

Techniques: Knockdown, Staining, TUNEL Assay

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 sections were blocked with 5% goat serum for 1 h, followed by an overnight incubation at 4°C with the antibodies against DVL1 (1:100, 13–706, ProSci, Poway, California, United States), β- catenin (1:500, ab32572, Abcam), and SP2 (1:100, PA5- 103254, Thermo Fisher Scientific).

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 5 | SP2, poorly expressed in OA, represses DVL1 transcription. (A) Transcription factor binding sites near the mouse DVL1 promoter region predicted using the JASPAR Transcription Factors plugin from the UCSC Genome Browser. (B) Intersections of the predicted transcription factors and the significant DEGs obtained from high-throughput sequencing. (C) Positive staining of SP2 in the joint cartilage determined using IHC. (D) SP2 mRNA expression in the joint cartilage determined using RT-qPCR. (E) Binding between SP2 and the DVL1 promoter in chondrocytes determined using ChIP-qPCR assay; the extracted chondrocytes were administered lentiviral vectors carrying OE-NC or OE-SP2. (F) mRNA and (G) protein level of SP2 in mouse chondrocytes determined using RT-qPCR and WB analysis. (H) mRNA expression of DVL1 in mouse chondrocytes analyzed using RT-qPCR. (I) Regulation of SP2 on transcription activity of the DVL1 promoter in chondrocytes determined using the dual lucifer- ase reporter gene assay. For animal experiments, each group contained five mice. For cell experiments, three biological replicates were performed. Differences were compared by the unpaired t-test (C–I).

Article Snippet: The sections were blocked with 5% goat serum for 1 h, followed by an overnight incubation at 4°C with the antibodies against DVL1 (1:100, 13–706, ProSci, Poway, California, United States), β- catenin (1:500, ab32572, Abcam), and SP2 (1:100, PA5- 103254, Thermo Fisher Scientific).

Techniques: Binding Assay, Next-Generation Sequencing, Staining, Expressing, Quantitative RT-PCR, ChIP-qPCR, Activity Assay, Reporter Gene Assay

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 6 | DVL1 upregulation aggravates cartilage injury in mice alleviated by SP2. DMM-challenged mice were introduced with adenoviral vectors carrying OE-NC/OE-SP2 alone or with the additional OE-NC/OE-DVL1. (A) mRNA expression of SP2 and DVL1 in the joint cartilage deter- mined using RT-qPCR. (B) Positive staining of SP2 and DVL1 in the joint cartilage determined using IHC. (C) Protein levels of β-catenin in the joint cartilage determined using WB analysis. (D) Cartilage morphology in the mouse knee joints determined using Safranin O/fast green staining. (E) Positive TRAP staining of the mouse knee joints. (F) Protein levels of COL10A1 and MMP13 in the mouse cartilage determined using WB analysis. Each group contained five mice. Differences were compared by ANOVA (A–F).

Article Snippet: The sections were blocked with 5% goat serum for 1 h, followed by an overnight incubation at 4°C with the antibodies against DVL1 (1:100, 13–706, ProSci, Poway, California, United States), β- catenin (1:500, ab32572, Abcam), and SP2 (1:100, PA5- 103254, Thermo Fisher Scientific).

Techniques: Expressing, Quantitative RT-PCR, 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 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 sections were blocked with 5% goat serum for 1 h, followed by an overnight incubation at 4°C with the antibodies against DVL1 (1:100, 13–706, ProSci, Poway, California, United States), β- catenin (1:500, ab32572, Abcam), and SP2 (1:100, PA5- 103254, Thermo Fisher Scientific).

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

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 8 | Schematic illustration of the mechanism. In cartilage tissues of osteoarthritic mice, decreased expression of SP2 resulted in its attenuated transcriptional repression of DVL1. Aberrantly expressed DVL1 activated the β-catenin signaling, leading to chondrocyte damage and osteoarthritis progression.

Article Snippet: The sections were blocked with 5% goat serum for 1 h, followed by an overnight incubation at 4°C with the antibodies against DVL1 (1:100, 13–706, ProSci, Poway, California, United States), β- catenin (1:500, ab32572, Abcam), and SP2 (1:100, PA5- 103254, Thermo Fisher Scientific).

Techniques: Expressing