Fig. 4: Reduced CpG hydroxymethylation at the E2F1 binding motif contributes to ESRP1 downregulation under hypoxia. | Oncogenesis

Fig. 4: Reduced CpG hydroxymethylation at the E2F1 binding motif contributes to ESRP1 downregulation under hypoxia.

From: E2F1 and epigenetic modifiers orchestrate breast cancer progression by regulating oxygen-dependent ESRP1 expression

Fig. 4

A hMeDIP of DNA isolated from MCF7 cells (Normoxia versus Hypoxia) using 5 hydroxymethylcytosine antibody followed by qRT-PCR, relative to input and control IgG (n = 3). B Immunoblots of ESRP1 after bobcat (70–90 μM) treatment under normoxia in HCC1806. C Densitometric analysis of representative blots. D hMeDIP in HCC1806 after bobcat (70–90 μM) treatment under normoxia, followed by qRT-PCR relative to input and control IgG (n = 3). E hMeDIP in MCF7 cells transfected with shRNA against TET1, TET2, TET3 versus shcontrol cells under normoxia, followed by qRT-PCR relative to input and control IgG (n = 3). F MeDIP in MCF7 cells transfected with shRNA against TET1, TET2, TET3 versus shcontrol cells under normoxia, followed by qRT-PCR relative to input and control IgG (n = 3). G Immunoblots of TET1, TET2, TET3, and ESRP1 protein expression in shTET1, shTET2, shTET3, and shcontrol MCF7 cells under normoxic condition. H, I Densitometric analysis of representative blots compared to shControl normalized to one. J ChIP qRT-PCR on ESRP1 promoter using E2F1 antibody in TET3 knockdown MCF7 cells under normoxia. Fold enrichment (E2F1/IgG) was normalized to 5% input. K hMeDIP in normal and breast tumor tissue genomic DNA and qRT-PCR of ESRP1promoter region relative to input and control IgG (n = 14). Error bars show mean values ± SD (n = 3 unless otherwise specified) calculated using two-tailed Student’s t test, ns (non-significant), **P < 0.01 and ***P < 0.001.

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