Fig 4.

Expression of GPC1 inactivates the G₁/S checkpoint. (A) E2F transactivation reporter assay. U87-MG cells were cotransfected with the E2F reporter pGL2(E2F)2 and the internal control pGL4.70 and transduced subsequently with different doses of 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. Each bar represents the mean ± SE from three independent experiments. (B and C) U87-MG cells were infected with different doses of control or GPC1 adenovirus for 48 h as indicated. (B) Immunoblotting for pRb on low (6%)- and high (12%)-concentration SDS-polyacrylamide gels and for p130 and p107 on a 6% SDS-polyacrylamide gel. β-Actin was used as a loading control. (C) Quantitative RT-PCR analysis of pRB mRNA. Total RNAs were isolated from the adenovirus-infected cells. Relative pRb mRNA levels, comparing GPC1 and control adenovirus-treated cells, were measured by quantitative RT-PCR. Each bar represents the mean ± SE from three independent experiments. The increase in E2F transactivation was GPC1 dose dependent, and pRb, p130, and p107 proteins were downregulated by expression of GPC1 in a dose-dependent pattern, suggesting inactivation of the G₁/S checkpoint.