Figure 6.

p130Cas inhibits TGF-β–mediated transcription and growth arrest. (A) MEFs transfected with the Smad3-responsive reporter (CAGA)₁₂-luc were incubated with or without 2 ng/ml TGF-β. Luciferase activity was measured 36 h later and normalized to the β-galactosidase activity. Luciferase activity is expressed as the mean ± SD of triplicates from a representative experiment performed at least three times. *p < 0.01 versus Cas+/+ MEFs. (B) MEFs transfected with the indicated plasmids were incubated with or without 2 ng/ml TGF-β, after which luciferase activity was measured as described in A. Data are representative of at least three independent experiments, and they are expressed as the mean ± SD. *p < 0.01 versus Cas+/+ MEFs. (C) HEK293T cells were cotransfected with a Smad3-responsive luciferase reporter, Smad3, and/or p130Cas, as indicated, after which luciferase activity was measured as described in A. Luciferase activity is expressed as the mean ± SD of triplicates from a representative experiment performed at least three times. *p < 0.05 and **p < 0.01. (D) HaCaT cells treated with the indicated concentration of TGF-β were subjected to immunoblot analysis with anti-p21 mAb and anti-p15 Ab. (E) HaCaT cells transfected with control or p130Cas siRNA were treated with 2 ng/ml TGF-β, and then they were subjected to immunoblot analysis with anti-p21 mAb and anti-p15 Ab. (F) HaCaT cells, (G) Cas+/+ and Cas−/− MEFs were incubated with or without the indicated concentration of TGF-β, after which [³H]thymidine incorporation was assayed as described in Materials and Methods. [³H]thymidine incorporation by cells incubated without TGF-β was defined as 100%. Data are representative of four independent experiments, and they are expressed as means ± SD. *p < 0.05 and **p < 0.01 versus HaCaT-vector or Cas+/+ MEFs.