EBNA3C expression leads to E2F1 destabilization via an ubiquitin-proteasome dependent pathway.

<p>A) HEK 293 cells were co-transfected with flag-E2F1 and either vector control (lanes 1 and 3) or myc-EBNA3C (lanes 2 and 4) with GFP expressing vector. At 36 h posttransfection, samples were treated with either 20 µM MG132 (+lanes) or DMSO (−lanes) for 6 h and resolved by 10% SDS-PAGE and probed with the indicated antibodies. B) HEK 293 cells were similarly transfected with expression plasmids for flag-E2F1, myc-tagged EBNA3C and GFP plasmids as indicated. At 36 h post-transfection, cells were treated with 40 µg/ml cyclohexamide (CHX) for indicated lengths of time. 5% of the lysate from each sample were resolved by 9% SDS-PAGE and western blotted with indicated antibodies. C) 15 million HEK 293 cells transfected with different combinations of expression plasmids as indicated. Cells were harvested at 36 h, and total protein was immunoprecipitated (IP) with flag antibody and samples were resolved by 9% SDS-PAGE. D) Approximately 10 million of stably generated LCLs with either Sh-control (Sh-Con) or Sh-EBNA3C (Sh-E3C) were incubated with 100 µg/ml CHX for indicated lengths of time in RPMI medium containing either 10% FBS (+serum/DMSO) or 0.1% FBS plus 5 µM etoposide (−serum/+Etop) at 37°C. 10% of the lysate from each sample were resolved by 9% SDS-PAGE and western blotted with indicated antibodies. E) Approximately 50 million LCLs with either control Sh-RNA (Sh-Con) or EBNA3C directed Sh-RNA (Sh-E3C) were harvested after 10 h incubation with proteasome inhibitor MG132 (40 µM). Cells were lysed and E2F1 was immunoprecipitated (IP). Samples were resolved by 9% SDS-PAGE and western blotting (WB) was done by stripping and reprobing the same membrane. F) Saos-2 cells transfected with the WT E2F1 reporter plasmid in the presence of either flag-E2F1 alone or flag-E2F1 plus myc-EBNA3C expressing plasmids followed by incubated with either DMSO or MG132 (20 µM) were subjected for reporter assay as essentially described in ‘<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002573#s2" target="_blank">Materials and Methods</a>’. Mean values and standard deviations of three independent experiments are presented. Bottoms panels indicate a representative blot of 5% of the total cell lysates resolved by appropriate % SDS-PAGE demonstrating the expression levels of ectopically expressed proteins. E2F1 protein bands were quantified using Odyssey imager software as indicated as either bar diagrams (A, B and D) or arbitrary numerical values (relative intensity, RI) at the bottom of gel (B–E) based on GFP or GAPDH loading controls, where applicable. G–H) Approximately 10 million of indicated cells incubated with either DMSO or MG132 (40 µM) for 10 h, harvested and 10% of total lysates were subjected for western blots with indicated antibodies. For all western blots, where appropriate, GAPDH serves as an internal loading control and GFP as a transfection efficiency control. Western blotting was done by stripping and reprobing the same membrane. Protein bands were quantified using Odyssey imager software as indicated either as arbitrary numerical values at the bottom of gel or as bar diagrams based on either GAPDH or GFP loading control. I) In response to DNA damage signals E2F1 gets stabilized and transcriptionally activates pro-apoptotic genes p73 and Apaf-1, which eventually induces apoptosis. In EBV transformed cells, by forming a stable complex with E2F1, EBNA3C inhibits its DNA binding activity and inhibits the transcriptional activation of p73 and Apaf-1. Moreover, EBNA3C specifically targets E2F1 for an ubiquitin-proteasome mediated degradation, which altogether blocks apoptotic induction. Moreover, sh-RNA designed against E2F1 showed reverse consequences and augments the apoptotic resistance of the cells.