Supplementary Figure S1 A-E from Drugging MYCN Oncogenic Signaling through the MYCN-PA2G4 Binding Interface

Abstract

<p>Supplementary Figure S1. PA2G4 is a MYCN transactivation target gene. A, Quantification of protein expression using anti-PA2G4 and anti-MYCN antibodies against whole cell protein lysates from BE(2)-C and Kelly cells, following MYCN siRNA knockdown for 48 hours. B, mRNA expression of PA2G4 and MYCN in SH-EP MYCN3 overexpression cells treated with 1µg/ml doxycycline for 24-96 hr. C, The effect of doxycycline-induced MYCN overexpression in SHEP-TRE-MYCN cells on c-MYC and PA2G4 protein expression. D, Chromatin immunoprecipitation (ChIP) assay in Kelly cells using an anti-MYCN antibody, and real-time PCR analysis with primers identifying the MYCN DNA binding sites in the PA2G4 gene promoter (500bP upstream of transcription start site [TSS]) or Intron 1a & 1b regions of the PA2G4 gene, with and without MYCN siRNA knockdown. ChIP and real-time PCR analysis using primers against a region 1200bp upstream of TSS was used as a negative control for MYCN chromatin binding. ChIP and real-time PCR analysis using primers against the ornithine decarboxylase (ODC1) gene promoter region was used as a positive control for MYCN chromatin binding. E, Immunoblot analysis of PA2G4, MYCN and MYC protein levels in a panel of human MYCN amplified and non-amplified neuroblastoma, and normal fibroblast, cell lines using antibodies recognising PA2G4, MYCN and MYC.</p>