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1.
Figure 4

Figure 4. From: Smurf2 Induces Degradation of GSK-3? and Upregulates ?-Catenin in Chondrocytes: A Potential Mechanism for Smurf2-Induced Degeneration of Articular Cartilage.

Upregulation of β-catenin protein levels in Col2a1-Smurf2 articular cartilage. (A–D) Examination of β-catenin levels in 2.5 week-old WT (A, C) and Col2a1-Smurf2 (B, D) femoral articular cartilage by immunofluorescence (A, B) and in situ hybridization (C, D). The regions between the two blue dotted lines in (C, D) represent articular cartilage. (E–F) Examination of β-catenin in 8 month-old WT (E) and Col2a1-Smurf2 (F) articular cartilage by immunohistochemistry. Red arrows and yellow arrowheads in (F) indicate β-catenin positive articular chondrocytes undergoing maturation and endochondral ossification during osteophyte formation, respectively. The bar in (A) represents 50 µm for (A–F).

Qiuqian Wu, et al. Exp Cell Res. ;315(14):2386-2398.
2.
Figure 6

Figure 6. From: Smurf2 Induces Degradation of GSK-3? and Upregulates ?-Catenin in Chondrocytes: A Potential Mechanism for Smurf2-Induced Degeneration of Articular Cartilage.

Interaction of Smurf2 with GSK-3β induces its ubiquitination and proteasomal degradation in murine sternal chondrocytes. (A–B) Interaction between ectopic human Smurf2 and endogenous GSK-3β in Col2a1-Smurf2 sternal chondrocytes (A) and between transiently overexpressed human Smurf2 with endogenous GSK-3β in WT sternal chondrocytes (B). Cell lysates were subjected to immunoprecipitation (IP) with an anti-hSmurf2 antibody that does not cross-react with mouse Smurf2 (A, upper panel), or with other antibodies as indicated in (B, upper panel). (C–D) Interaction of Smurf2 with GSK-3β induces its ubiquitination. The cell lysates used in (A) were subjected to IP with the anti-GSK-3β followed by a blotting with an anti-ubiquitin antibody (C, upper panel), and the cell lysates used in (B) were subjected to IP with the anti-GSK-3β or an anti-pSmad2 antibody followed by a blotting with the anti-ubiquitin (D, upper panel). (E) GSK-3β protein decay assay. Murine sternal chondrocytes were treated with cycloheximide for 30– 120 minutes as indicated. Cell lysates from each time point were subjected to immunoblotting with the anti-GSK-3β antibody. (F) The protein levels of GSK-3β in (E) were quantitated by scanning densitometry (normalized β-actin).

Qiuqian Wu, et al. Exp Cell Res. ;315(14):2386-2398.
3.
Figure 1

Figure 1. From: Smurf2 Induces Degradation of GSK-3? and Upregulates ?-Catenin in Chondrocytes: A Potential Mechanism for Smurf2-Induced Degeneration of Articular Cartilage.

Deep articular cartilage abnormality in postnatal Col2a1-Smurf2 mice. (A–B) Safranin O/fast green (SO/FG) staining of knee joints from 2.5 week-old WT (A) and Col2a1-Smurf2 (B) mice. (A’, B’) Enlarged images of boxed areas in (A, B). Green lines in (A’, B’) mark the thickness of deep articular cartilage. C, cell number; T, thickness of deep articular cartilage. Data are expressed as the mean ± SD. Statistical significance is assessed by an unpaired Student’s t-test (*p<0.05, n=6). (C–F) In situ hybridization of knee joints from 2.5 week-old WT (C, E) and Col2a1-Smurf2 mice (D, F) with 35S-UTP labeled riboprobe Col2 probe (C, D) and ColX probe (E, F). Yellow and white arrows in (E, F) indicate the ColX expression domain in the basal layer of femoral and tibial articular cartilage, respectively. The bar in (A) represents 200 µm for (A–F). The bar in (A’) represents 50 µm for (A’-B’).

Qiuqian Wu, et al. Exp Cell Res. ;315(14):2386-2398.
4.
Figure 3

Figure 3. From: Smurf2 Induces Degradation of GSK-3? and Upregulates ?-Catenin in Chondrocytes: A Potential Mechanism for Smurf2-Induced Degeneration of Articular Cartilage.

Decrease in cartilage matrix synthesis by Col2a1-Smurf2 articular chondrocytes. (A–D) SO/FG staining of knee joints from WT (A, C) and Col2a1-Smurf2 (B, D) mice at 2 months (A, B) and 4.5 months (C, D) of age. (A’-D’) Images at a higher magnification of boxed areas in (A-D). Arrows in (A’, C’) indicate the WT articular chondrocytes which are sparse and synthesizing high levels of negatively charged proteoglycans (red), while arrows in (B’, D’) indicate the maturing transgenic articular chondrocytes, in which the charged proteoglycan content is decreased. Asterisks mark the extracellular matrix. (E–H) Analysis of Col2 expression levels in femoral articular cartilage of WT (E, G) and Col2a1-Smurf2 (F, H) mice by in situ hybridization (E, F) and immunohistochemistry (G, H). Arrows in (E, F) indicate the levels of Col2 mRNA, and arrows in (G, H) indicate the levels of Col2 protein. The bar in (A) represents 100 µm for (A–D). The bar in (A’) represents 50 µm for (A’-D’). The bar in (E) represents 150 µm for (E–H).

Qiuqian Wu, et al. Exp Cell Res. ;315(14):2386-2398.
5.
Figure 2

Figure 2. From: Smurf2 Induces Degradation of GSK-3? and Upregulates ?-Catenin in Chondrocytes: A Potential Mechanism for Smurf2-Induced Degeneration of Articular Cartilage.

Abnormal articular cartilage in Col2a1-Smurf2 mice. (A–B) Alcian blue, hematoxylin, and eosin (AHE) staining of 1 month-old WT (A) and Col2a1-Smurf2 (B) mouse tibia. (A’, B’) Enlarged images of boxed areas in (A, B). Contours mark the calcified articular cartilage. Arrows in (B’) indicate ectopic ossification in the calcified zone. (C–D) Alizarin red staining of 1 month-old WT (C) and Col2a1-Smurf2 (D) mouse tibia. Contours mark the calcified region of articular cartilage. (E–H) AHE staining of WT (E, G) and Col2a1-Smurf2 (F, H) mouse knee joints at 2 months (E, F) and 4.5 months (G, H) of age. (F’-H’) Images at a higher magnification of boxed areas in (F–H). Arrows in (F’, H’) mark clusters and column-like structures, respectively. Green lines in (E–H) indicate the thickness of articular cartilage. Data are expressed as the mean ± SD. Statistical significance is assessed by an unpaired Student’s t-test (*p<0.05, n=6). The bar in (A) represents 100 µm for (A, B, and E-H). The bar in (A’) represents 50 µm for (A’, B’, C, D, and E’-H’).

Qiuqian Wu, et al. Exp Cell Res. ;315(14):2386-2398.
6.

Figure 5. From: Smurf2 Induces Degradation of GSK-3? and Upregulates ?-Catenin in Chondrocytes: A Potential Mechanism for Smurf2-Induced Degeneration of Articular Cartilage.

Upregulation of β-catenin protein levels and signaling in newborn Col2a1-Smurf2 chondrocytes through reduction of GSK-3β. (A–D) Examination of β-catenin levels in newborn WT (A, C) and Col2a1-Smurf2 (B, D) tibia by immunofluorescence (A, B) and in situ hybridization (C, D). Arrows in (A) indicate the perichondrium surrounding the proliferating (p) and hypertrophic (h) chondrocytes. (A’, B’) Enlarged images of boxed areas in (A, B). White arrowheads and yellow arrowhead in (B’) indicate the nucleus with high β-catenin, and the cytoplasm with low β-catenin, respectively. (E-G) Examination of β-catenin levels and signaling in newborn sternal chondrocytes by Western blotting (E), real time RT-PCR (F) and TOPflash assay (G). Data in (G) is expressed as the mean ± SD. Statistical significance is assessed by an unpaired Student’s t-test (**p<0.01, n=3). (H, I) Examination of the protein levels of Axin (H) and GSK-3β (I) in newborn sternal chondrocytes by Western blotting. (I’) The protein levels GSK-3 were quantitated by scanning densitometry (normalized β-actin). Data is expressed as the mean ± SD of three independent experiments. Statistical significance is assessed by an unpaired Student’s t-test (**p<0.01). The bar in (A) represents 200 µm for (A–D). The bar in (A’) represents 25 µm for (A’-B’).

Qiuqian Wu, et al. Exp Cell Res. ;315(14):2386-2398.

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