Fig. 4. EED overexpression restores interaction of HOTAIR with PRC2 and reduces ZO-1 mRNA with genistein treatment. (a) 786-O and ACHN cells in 6-well plates were treated with 25 uM genistein for 24 hours and transfected with 100 ng of control vector (pcMV6-ENTRY) or EED expression vector and incubated for 72 hours in the presence of genistein. PRC2 from 786-O and ACHN cells cultured with genistein (25 µM) or control for 96 hours were pulled down using anti-EZH2 antibody or control IgG. HOTAIR associated with the PRC2 was detected by quantitative RT-PCR. Data from quantitative RT-PCR are presented with respect to control IgG that is set to a value of 1 *p<0.05, **p<0.01. (b) Immunoprecipitated PRC2 subunits were detected by Western blot. (c) EED expression was analyzed by Western blot. D. ZO-1 mRNA expression level was measured by quantitative RT-PCR.