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. 2013 Sep 2;33(30):3927–3938. doi: 10.1038/onc.2013.361

Figure 4.

Figure 4

A subset of EWS-FLI1-repressed genes is regulated by FOXO1. (a) Venn Diagram showing the intersection between EWS-FLI1-repressed and FOXO1-activated genes. Silencing of reactivated endogenous FOXO1 under EWS-FLI1 knockdown conditions prohibited the activation of 94 EWS-FLI1-repressed genes, whereas the overexpression of nuclear FOXO1 mutants (Flag-FOXO-T24A/S256A/S319A(AAA) (AKT-p-resistant), Flag-FOXO-S249A/S29A (CDK2-p-resistant) led to reactivation of 94 and 58 EWS-FLI1-repressed genes, respectively. (b, c) Knockdown of reactivated endogenous FOXO1 was used to demonstrate the dependency of EWS-FLI1-mediated repression on FOXO1 suppression in two different ES cell lines. The mRNA expression was normalized to pCDNA3 empty vector control expression or to sh-scrambled control without doxycycline treatment or sh-RNA targeting EWS-FLI1 (shEF), and statistical relevance was analyzed using the unpaired t-test or one-sample t-test, respectively. B2M was used as internal control and doxycycline was applied for 72 h. (d) Representative FOXO1 protein expression corresponding to b, showing specificity and efficiency of sh-RNA-mediated FOXO1 silencing (shRNA no. 3). β-Actin was used as loading control. (e) Representative western blot for protein expression of endogenous FOXO1 upon inducible EWS-FLI1 knockdown as well as ectopically expressed wild-type and nuclear FOXO1 mutant, corresponding to (f) showing similar levels of ectopically expressed and induced endogenous FOXO1. (f, g) Validation of EWS-FLI1-repressed genes that can be reactivated by nuclear FOXO1 in two ES cell lines. A673sh and TC252 cells were transfected with AKT- or CDK2-phosphorylation-resistant versions of Flag-FOXO1, and mRNA expression of three genes representative of the overlap between EWS-FLI1-repressed and FOXO1-activated genes was measured by reverse transcription–quantitative PCR (RT-qPCR). Results represent the mean±s.e.m. of three experiments performed. *P<0.05 and **P<0.01. EPAS1, endothelial PAS domain protein 1 (entrez gene number: ID: 2034); MME, membrane metallo-endopeptidase (ID: 4311); OSMR, oncostatin M receptor (ID: 9180)