Figure 1

Ectopic E2F1 expression directly upregulates ERIC RNA levels. A) U2OS and H1299 cells containing conditionally active E2F1 were induced to activate E2F1 by OHT (times indicated). RNA was extracted and RNA deep-sequencing analysis was employed. FI- fold of increase in RNA levels after E2F1 induction, determined by RNA sequencing. B) U2OS, H1299, WI38 and SAOS-2 cells containing conditionally active E2F1 were induced to activate E2F1 by addition of OHT (times indicated). RNA was extracted, and ERIC RNA levels determined by Real- time RT-PCR and normalized to GAPDH. U2OS cells containing an empty vector (vec) served as a control. C) U2OS cells were transfected with an empty vector (Vec) or a vector expressing either wild type E2F1 (E2F1) or mutant E2F1 (E2F1 mut). Upper panel- RNA was extracted and ERIC RNA levels determined by Real-time RT-PCR and normalized to GAPDH. Lower panel- Proteins were extracted and western-blot analysis performed using antibodies directed against E2F1 and GAPDH. D) Induction of ERIC by E2F1 is p53-independent. U2OS cells stably expressing ER- E2F1 were infected with either a retrovirus expressing non specific shRNA (shNS) or a retrovirus expressing shRNA directed against p53 (shp53). Cells were left untreated (-) or incubated with OHT (100nM) for 16 hr (+). Upper panel- RNA was extracted and ERIC RNA levels were determined by real-time RT-PCR and normalized to GAPDH. Lower panel- Proteins were extracted and western-blot analysis was performed using antibodies directed against p53 and Actin. E) Induction of ERIC does not require protein synthesis. U2OS cells expressing ER-E2F1 were left untreated or incubated with OHT (100 nM, 8 hr); cells were then treated or not with 10 μg/mL cycloheximide (CHX) for 8 h. RNA was extracted and ERIC RNA levels determined by real-time RT-PCR and normalized to GAPDH. The average of two independent experiments is presented.