Fig 2: NRG1 is a critical transcriptional effector that is necessary and sufficient for BRD4-mediated transformation.(A) Two experimental schemes used to identify BRD4 transcriptional effectors. (B) Venn diagram illustrating mRNA transcripts > 2FC downregulated by AZD5153 in OV0857F tumors, and mRNA transcripts > 2FC upregulated by either short- or long-BRD4 expression in IOSE cells. (C) qPCR for NRG1 mRNA performed on BRD4 expressing IOSE cells. (D) Immunoblots confirming modulation of RTK signaling from BRD4 expressing IOSE cells. (E) ERBB2 GSEA profile is enriched in BRD4-amplified HGSOC TCGA patients. (F-G) ChIP-qPCR studies of BRD4 binding to NRG1 TSS or putative enhancers in IOSE cells expressing either the long- or short-isoform of BRD4. Cells were treated with 200 nM AZD5153 for 4 hr and ChIP was performed with antibodies against either FLAG or IgG. Percentage enrichment was calculated over total input DNA. Data represent average ± SEM of three replicates. (H-I) qPCR analysis of MYC and NRG1 levels in IOSE cells overexpressing BRD4 isoforms after 200 nM AZD5153 treatment for indicated periods of time. All expression levels are normalized to GAPDH and presented as fold increase relative to empty-vector expressing cells. (J) NRG1 knockdown confirmed by immunoblot in BRD4-expressing IOSE cells transduced with a scramble control or shNRG1 lentiviral vectors. (K-L) BRD4-IOSE cells expressing shNRG1 vectors were plated in soft agar to examine colony formation potential. Empty-vector IOSE cells were plated in soft agar +/- 10 ng/mL recombinant NRG1.