Figure 4

FOXM1 and ERK2 co-occupy genomic loci of cluster C1 genes, and impact of knockdown of FOXM1, or ERK1, or ERK2, or treatment with FOXM1 inhibitor. (A) UCSC Browser location of the binding sites we identified for FOXM1, estrogen receptor α and extracellular signal-regulated kinase 2 (ERK2) for four representative genes in the C1 cluster (SIRT1, B-Myb, CHEK1 and ABCG2). (B) Western blot showing ERK1, ERK2 and FOXM1 levels after small interfering RNA (siRNA) knockdown of ERK1 or ERK2. (C) Western blot showing FOXM1 and phosphorylated mitogen-activated protein kinase levels after alternate reading frame (ARF) (FOXM1 inhibitor) treatment. (D) FOXM1 recruitment to chromatin sites was assessed after ARF or extracellular signal-regulated kinase kinase 1 (MEK1) inhibitor treatment and followed by quantitative RT-PCR (qRT-PCR). (E) Chromatin immunoprecipitation (ChIP) for ERK2 was assessed after ARF or control vehicle treatment. (F) ChIP-reChIP showing binding site co-occupancy by FOXM1 and ERK2. Immunoprecipitation was done first for FOXM1 and then for ERK2. (G) Levels of representative C1 genes in siCtrl- or siFOXM1-treated cells or in cells with ARF treatment or overexpression of FOXM1. (H) Western blot showing FOXM1 levels after siRNA or ARF treatment or overexpression. (I) MEK1 inhibitor blocks tamoxifen (TAM) stimulation of FOXM1 target genes. Cells were pretreated with 10 μM MEK1 (AZD6244) or vehicle for 45 minutes and then treated with vehicle (0.1% EtOH) or 1 μM TAM in the presence or absence of inhibitor for 4 hours. RNA was isolated and qPCR analysis was done.