Figure 2

Endogenous E2F1 regulates expression of ERIC. A) WI38 cells were infected with a retrovirus expressing either wild-type E7 (E7) or an RB-binding–deficient E7 mutant (E7-mut). Upper panel-RNA was extracted and ERIC RNA levels determined by Real-time RT-PCR and normalized to GAPDH levels. Lower panel- proteins were extracted and western blot analysis performed using antibodies directed against E2F1, CCNE1 and Actin. B) Upper panel- U2OS cells were transfected with either a nonspecific siRNA (siNS) or an siRNA directed against E2F1 (si#1 and si#2). RNA was extracted and ERIC RNA levels determined by Real-time RT-PCR and normalized to GAPDH levels. Lower panel- Proteins were extracted from cells and western blot analysis performed using antibodies directed against E2F1 and actin. C) Upper panel- a schematic representation of the human ERIC promoter. The E2F-binding site is represented as 8-mer nucleotide sequences. The transcription start site (+1) is indicated by an arrow. The DNA fragment amplified by RT-PCR is represented by arrows (-523/-10). Lower panel- Chromatin Immuno Precipitation analysis was conducted using U2OS cells. Cross-linked chromatin was precipitated using antibodies specific to E2F1 or IgG. Then, ERIC and Actin promoter fragments were amplified by RT-PCR. Input DNA represents 0.5% of total chromatin.