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. Author manuscript; available in PMC: 2012 Jan 9.

Published in final edited form as: Mol Cancer Res. 2010 Aug 24;8(10):1375–1387. doi: 10.1158/1541-7786.MCR-09-0537

Restoration of Smad3 transcriptional activity with siRNA knockdown of CDK4. (A) MCF7 cells were transfected with scrambled (SC) or cdk4 siRNA (siRNA) for 48 hours, then the cells were lysed and the level of CDK4 protein was determined in untransfected cells (UC) and transfected cells by immunoblot analysis using anti-CDK4 antibody. GAPDH was used as loading control. (B) MCF7 study cell lines were co-transfected with the Smad-responsive CAGA-luc reporter construct and Renilla luciferase reporter; siRNA or cdk4 siRNA; and either empty vector, wild type (WT) Smad3, or T178 Smad3 mutant expression vectors. Firefly and Renilla luciferase activities were determined. Data are shown as fold increase in normalized luciferase activity (firefly/Renilla) compared with empty vector-transfected MCF7 or T47D cells. Error bars indicate standard deviation from the mean of normalized luciferase activity for each study condition.

Restoration of Smad3 transcriptional activity with siRNA knockdown of CDK4. (A) MCF7 cells were transfected with scrambled (SC) or cdk4 siRNA (siRNA) for 48 hours, then the cells were lysed and the level of CDK4 protein was determined in untransfected cells (UC) and transfected cells by immunoblot analysis using anti-CDK4 antibody. GAPDH was used as loading control. (B) MCF7 study cell lines were co-transfected with the Smad-responsive CAGA-luc reporter construct and Renilla luciferase reporter; siRNA or cdk4 siRNA; and either empty vector, wild type (WT) Smad3, or T178 Smad3 mutant expression vectors. Firefly and Renilla luciferase activities were determined. Data are shown as fold increase in normalized luciferase activity (firefly/Renilla) compared with empty vector-transfected MCF7 or T47D cells. Error bars indicate standard deviation from the mean of normalized luciferase activity for each study condition.