Figure 4.

E2F1 up‐regulates the expression of endogenous Siah1. (A) H1299 cells were incubated in medium containing 0.5% serum for 48 hrs Treated cells were then transfected with either pcDNA3 (200 ng) or pcDNA3‐E2F1 (200 ng). Thirty‐six hours after transfection, cells were harvested and subject to Western analysis with indicated antibodies. (B) H1299 cells were treated under same conditions as described in (A). Thirty‐six hours after transfection, cells were harvested for RNA isolation and equal amounts of total RNA were reverse transcribed and amplified. PCR amplification of β‐actin was performed simultaneously as negative control. Total RNA used for each PCR reaction was shown as loading control (bottom panel). (C) H1299 cells were transfected with 1ug of E2F1 shRNA construct pU6‐E2F1 or pU6 empty vector. Forty‐eight hours after transfection, cells were harvested for preparation of total RNA and followed by semi‐quantitative RT‐PCR analysis with primer pairs specific to E2F1, Siah1 and β‐actin. (D) H1299 cells were treated the same way as described in (C) and then subject to Western analysis using antibodies against E2F1, Siah1 and β‐actin, respectively. (E) H1299 cells were incubated in medium containing 0.5% serum for 48 hrs and then released in medium containing 20% serum. Cells were harvested at the indicated time‐points and applied to Western blots and FACS analysis.