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Fig. 5 | Journal of Hematology & Oncology

Fig. 5

From: ETV4 promotes late development of prostatic intraepithelial neoplasia and cell proliferation through direct and p53-mediated downregulation of p21

Fig. 5

ETV4 binds and downregulates the CDKN1A promoter. a Putative ETV4-binding sites (ETV4 BS) on CDKN1A promoter are indicated by a diamond. The numbers indicate the position of ETV4 BS relative to the transcription start site (TSS). The localization of the primers for the qPCR is indicated. b qPCR analysis of ChIP performed on RWPE cells transiently transfected with either the ETV4-Flag expressing vector (ETV4) or a vector expressing only Flag (CTL), the chromatin was immunoprecipitated using the anti-flag antibody (CTL-Flag and ETV4-Flag) or IgG (CTL-IgG and ETV4-IgG) as controls. The signals obtained from the ChIP are expressed as percentage of the imput sample. ETV4-binding sites (ETV4 BS-A, BS-B, BS-C) as shown in a. Positive control: COX2 ETV4-binding site. Negative controls: a fragment from G6PD gene. c Diagram of the human CDKN1A promoter (top) and of the luciferase vector (ETV4-BS-A) containing the 845 bp region upstream the CDKN1A TSS (bottom). Diamonds indicate ETV4 BS. d Quantification of dual luciferase reporter assay in RWPE and PNT1A cells transiently transfected with vectors in which firefly luciferase expression is driven by the 865 bp CDKN1A promoter fragment (see above) containing either the wild-type (ETV4-BS-A_wt) or the mutant (ETV4-BS-A_mut) ETV4 BS-A. The bar diagram shows the relative luciferase activity (Firefly/Renilla ratio) from cells transfected with an ETV4-expressing vector (+ETV4, grey, or striped bars) normalized to those transfected with an empty vector (CTL, black bars). Data represent average and standard error of triplicate measurements of at least 3 independent experiments. e Quantification of dual luciferase reporter assay in PC3 cells transiently transfected with luciferase vectors containing either the wild-type (ETV4-BS-A_wt) or the mutant (ETV4-BS-A_mut) ETV4 BS-A (see above). The bar diagram shows the relative luciferase activity (Firefly/Renilla ratio) from cells with ETV4 silencing (–ETV4, grey, or striped bars) normalized to those without ETV4 silencing (CTL, black bars). ETV4 silencing has been obtained with either one of 2 ETV4-specific shRNA (shETV4a, shETV4b) or a siRNA against ETV4 (siETV4). Data represent average and standard error of triplicate measurements of at least 4 (shETV4) or 3 (siETV4) independent experiments. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001

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