Figure 2

Successful shRNA/cDNA targeting using designed entry vectors. A) pENTR constructs targeting exon 2 of p16 were transfected into 293T cells. RNA was isolated 48 hours post transfection and the mRNA expression level was determined using Q-RT-PCR. The average represents fold gene expression level relative to pENTRhU6hH1 transfected 293T cells (Students t-test; p < 0.01). B) pENTR constructs for CCND1 and/or CCND3 shRNA expression were recombined with plko.puro.DEST. Western blot shows the successful knockdown of CCND1/CCND3 in Panc1 cells. C) pENTR constructs for p16 shRNA expression were recombined with plko.YFP.DEST. Western blot shows p16 knockdown in H6c7 cells transfected with p16 shRNA constructs. D) pENTR.Cre mediated Cdk4 overexpression in 293T cells transiently transfected with pENTR.Cdk4.ON. The average represents fold gene expression level relative to control vector cell line f) Luciferase activity downregulation by Cre protein in stable HeLa cells overexpressing Luc2 mediated by plko.dsRed2.Luc2.ON lentivirus.