The Action Mechanism of Omomyc Figure 1. Omomyc binding specificity. A) Omomyc binds c-Myc and N-Myc. Immunoblotting (WB) with FLAG and ER antibodies – as indicated – of immunoprecipitations performed with FLAG antibodies on 293T cells transfected with FLAG-c-Myc or FLAG-N-Myc expressing vectors together with the Omomer expressing vector. B) Omomyc binds Max but not Mad and two representative HLH proteins. Immunoblotting (WB) with FLAG antibodies of GST pull-down assays performed with GST, GST-MAX, GST-MAD, GST-ID1 and GST-HEB (10 `ıg each) on 293T cells transfected with the FLAG-Omomyc expressing vector. 293T cells transfected with ID2 or FLAG-13I [65] expressing vectors were used as positive control for GST-HEB and GST-ID1, respectively. Extracts from bacteria expressing His-MAX were used as positive controls for GST-MAX and GST-MAD. C) Omomyc does not bind Hif-1a. Immunoblotting (WB) with Hif-1a, Max and FLAG antibodies – as indicated – of immunoprecipitations performed with FLAG antibodies on 293T cells transfected with Hif-1a, Max and FLAG-Omomyc expressing vectors. D) Omomyc binds Miz-1. Immunoblotting (WB) with Miz-1 and ER antibodies – as indicated – of immunoprecipitations performed with Miz-1 antibodies on 293T cells cotransfected with Miz-1 and Omomer expressing vectors. doi:10.1371/journal.pone.0022284.g001 Ectopic Myc expression triggered nuclear translocation of Miz-1 (about 40%) and formation of discrete subnuclear foci, as previously reported [30]. Miz-1 was partly relocated in the nucleus in the presence of an overexpressed Omomyc as well (about 35%). Endogenous Miz-1 – which is present in low amounts within cells [30] – will presumably be mostly in the nucleus in the presence of an overexpressed Myc or Omomyc. Altogether, these findings show that perturbation of the Myc interactome by Omomyc results not only from the inhibition of Myc binding to Max – which is required for E-box binding and transactivation – but also from the interaction of Omomyc with Miz-1, a Myc binding partner involved in transrepression. Omomyc affects the transcriptional response of fibroblasts to serum stimulation Myc proteins are able to bind to 10–20% of genomic loci and to modulate expression of hundreds to thousands of genes. Omomyc was shown to impair transactivation of the Myc activated target gene cad in Rat1 fibroblasts and not to affect downregulation of the Myc repressed target gadd45 [10]. To get an insight into widespread gene expression changes influenced by Omomyc, we analyzed the transcriptional response to serum stimulation of Rat1 fibroblasts stably infected with an Omomer producing or an empty retrovirus (Rat1-Omomer and Rat1-control cells, respectively, as described in [10]). c-Myc is known to be downregulated by serum starvation and sharply induced by serum addition – together with a number of other immediate early genes – reaching a peak after 1 to 2 h; c-Myc induction has a role in cell cycle re-entry [31–33]. Apart from cell proliferation, the serum response of fibroblasts integrates other processes – e. g. wound healing [34] – and serum regulated genes include genes that are regulated by Myc as well as genes that are not. Cells were serum-starved before serum stimulation and mRNA expression was analyzed at the time of serum re-addition (time 0) and 909 thereafter – a time point at which Myc induction is maximal – so that induction of a direct Myc target should closely follow the expression of Myc. mRNA PLoS ONE | www.plosone.org 3 July 2011 | Volume 6 | Issue 7 | e22284