C5.18 chondrocytes were transfected with β-TrCP with or without SMAD3 for 24 h. Cells were lysed, and Western blotting was performed using anti-β-catenin antibody. Transfection of β-TrCP significantly reduced the level of β-catenin, whereas cotransfection of SMAD3 and β-TrCP partially rescued β-TrCP-mediated β-catenin degradation.

a, C5.18 chondrocytes were cultured with or without TGF-β (1 ng/ml). After 4 h of TGF-β treatment, cells were fixed and incubated with either anti-β-catenin or anti-SMAD3 antibody, followed by incubation with either FITC- or TRITC-conjugated secondary antibody. The results showed that treatment with TGF-β induced the nuclear translocation of SMAD3 as well as β-catenin. b, similarly, treated cells were examined under a confocal microscope. In the absence of TGF-β, both β-catenin and SMAD3 stayed in the cytoplasm. Treatment with TGF-β promoted both SMAD3 and β-catenin nuclear translocation and co-localization.

a, primary chondrocytes isolated from β-catenin^flox/flox mice were infected with Ad-GFP or Ad-β-catenin for 24 h. After the medium was changed, cells were cultured in serum-free medium for 72 h. Deletion of the β-catenin gene in the β-catenin^flox/flox chondrocytes by Ad-Cre resulted in loss of responsiveness to TGF-β-induced cyclin D₁ protein levels. b, C5.18 chondrocytes were transfected with the constitutively active type I TGF-β receptor (caTβRI) with or without ICAT. Cells were lysed, and Western blotting was performed using anti-cyclin D₁ antibody. Transfection of the constitutively active type I TGF-β receptor alone greatly increased the protein level of cyclin D₁, whereas transfection of ICAT significantly reduced the constitutively active type I TGF-β receptor-induced up-regulation of cyclin D₁ protein. The empty vector transfection control is shown in the first lane.

a, the constitutively active β-catenin expression plasmid β-catenin^S33Y (β-cat^S33Y) was cotransfected with the TOPflash reporter into C5.18 chondrocytes. The luciferase activity was measured 24 h after transfection. Compared with the empty vector control (Vect), transfection of β-catenin^S33Y caused an ∼2-fold increase in reporter activity. b, primary chondrocytes isolated from TOPGAL transgenic mice were infected with Ad-β-catenin (Ad-β-Cat). Ad-GFP was used as a control. Ad-β-catenin infection resulted in a 2-fold increase in β-galactosidase (β-gal) activity in TOPGAL chondrocytes. TGF-β stimulated β-catenin signaling. c, TOPGAL chondrocytes were treated with different concentrations of TGF-β (0-10 ng/ml), and β-galactosidase activity was measured 24 h later. TGF-β increased β-galactosidase activity in a dose-dependent manner in TOPGAL chondrocytes. d, C5.18 chondrocytes were treated with TGF-(1 ng/ml) for different periods of time (0-480 min), and changes in β-catenin protein were examined by Western blotting. TGF-β up-regulated the protein levels of both total and active non-phosphorylated β-catenin. TGF-β significantly increased the β-catenin protein level within 15 min, and the maximum effect was reached at the 2-h time point.

a, C5.18 chondrocytes were synchronized in serum-free medium for 72 h. The cyclin D₁-luciferase (Luc) reporter construct was cotransfected with either the empty vector control (Vect) or β-catenin^S33Y (β-cat^S33Y) for 24 h, and luciferase assay was performed. Transfection of β-catenin^S33Y increased cyclin D₁ reporter activity by ∼5-fold compared with the empty vector control. b, C5.18 chondrocytes were infected with Ad-GFP or Ad-β-catenin (Ad-β-cat) for 24 h. After the medium was changed, cells were cultured in serum-free medium for 72 h. Western blotting showed that overexpression of β-catenin (Ad-β-catenin) increased the protein level of cyclin D₁ in C5.18 chondrocytes. c, C5.18 chondrocytes were synchronized in serum-free medium for 72 h. The cyclin D₁ reporter construct was cotransfected with either the empty vector control or ICAT for 24 h, and luciferase assay was then performed. Transfection of ICAT significantly reduced cyclin D₁ reporter activity. d, primary chondrocytes were isolated from β-catenin^flox/flox mice and their wild-type littermates and infected with Ad-Cre or Ad-GFP. Cell lysates were collected 48 h after infection. Infection with Ad-Cre almost completely abolished the protein expression of β-catenin in β-catenin^flox/flox chondrocytes. The protein level of cyclin D₁ was reduced when the β-catenin gene was deleted. In contrast, Ad-GFP had no effect on the protein levels of β-catenin and cyclin D₁.

a-d, BrdUrd labeling reagent was injected into the peritoneal cavities of 4-day-old neonatal mice. Mice were killed 4 h later, and hind limbs were harvested. The decalcified tissue sections were stained using a BrdUrd staining kit and counterstained with hematoxylin. a and c, wild-type mice; b and d, Smad3^-/- knock-out (KO) mice. e, morphometric analysis revealed that the number of BrdUrd-positive chondrocytes was significant lower in Smad3^-/- mice than in wild-type (Wt) mice. f, primary chondrocytes isolated from either wild-type or Smad3-deficient mice were cultured with BrdUrd reagent. After fixation, cells were incubated with FITC-conjugated anti-BrdUrd antibody. The results from flow cytometry showed that the numbers of BrdUrd-positive chondrocytes were reduced in Smad3^-/- mice with or without serum starvation. g, primary chondrocytes isolated from either wild-type or Smad3-deficient mice were lysed, and total RNA was extract. After reverse transcription, real-time PCR was performed using cyclin D₁ primers. The results showed that cyclin D₁ mRNA expression was decreased in Smad3^-/- chondrocytes.

a, primary chondrocytes were isolated from Smad3^-/- knock-out (KO) mice and their wild-type littermates. Expression of β-catenin protein was analyzed by Western blotting. Cell lysates containing 20 μg of protein were used for Western blotting. Compared with wild-type cells, Smad3-deficient chondrocytes showed lower protein levels of both total and active β-catenin. In addition, cyclin D₁ expression was also reduced in Smad3-deficient chondrocytes. b, wild-type and Smad3-deficient chondrocytes were transfected with the TOPflash reporter construct for 24 h. FOPflash (FOP) was used as control. TGF-β was added to cell cultures for 24 h prior to luciferase assay. The basal and TGF-β-induced TOPflash reporter activities were decreased in Smad3^-/- chondrocytes. The FOPflash reporter showed no response to TGF-β, confirming the specific effect of TGF-β on β-catenin signaling. c, the β-catenin^S33Y (β-cat^S33Y) plasmid was cotransfected with the TOPflash reporter into C5.18 chondrocytes in the presence or absence of SMAD3, and cell lysates were collected at 24 h for luciferase assay. β-Catenin^S33Y stimulated TOP-flash reporter activity, and SMAD3 slightly enhanced β-catenin^S33Y-induced TOPflash reporter activity. d, total cell lysates were harvested from C5.18 chondrocytes treated with or without TGF-β (1 ng/ml) for 1 h. Cell lysates were immunoprecipitated (IP) with anti-SMAD3 antibody, and Western blotting (WB) was performed using anti-β-catenin antibody (upper panels). Anti-His antibody was used as a control. The results demonstrate an interaction between SMAD3 and β-catenin in C5.18 chondrocytes. Endogenous β-catenin and SMAD3 protein expression was detected without immunoprecipitation in C5.18 chondrocytes (lower panels).