Fig 9.

Knockdown of Skp2, CDK2, and cyclin E significantly reduces the S phase fraction and diminishes the GPC1-induced cell cycle effect. (A) Effect of siRNA treatment on protein levels of Skp2, CDK2, and cyclin E (CCNE). U87-MG cells were transfected with 100 nM control (Ctrl) or gene-specific siRNAs against Skp2, CDK2, or CCNE for 24 h and then treated with control or GPC1 adenovirus at an MOI of 10 for 48 h. β-Actin was used as a loading control. (B) E2F-luciferase reporter assay. U87-MG cells were transfected with 100 nM control (Ctrl) or gene-specific siRNAs targeting Skp2, CDK2, or cyclin E (CCNE) for 24 h, transfected with pGL2(E2F)2 plus pGL4.70 for 6 h, and then treated with control or GPC1 adenovirus for 48 h. A dual-luciferase assay was performed, and the firefly luciferase activity from pGL2(E2F)2 was normalized to the Renilla luciferase activity from pGL4.70. (C and D) U87-MG cells were transfected with 100 nM control (Ctrl) or gene-specific siRNAs against Skp2, CDK2, or cyclin E (CCNE) for 24 h and treated with control or GPC1 adenovirus at an MOI of 10 for 48 h, and flow cytometry was performed after propidium iodide (PI) staining. The data were processed using ModFit LT 3.1 software. (C) Knockdown of these genes led to a significant reduction in the S phase fraction in the presence of control adenovirus. (D) GPC1-induced aneuploidy (i.e., DNA rereplication) was significantly reduced by knockdown of these genes. Each bar represents the mean ± SE from three independent experiments.