Fig. 6. Genistein reduces interaction of HOTAIR with ARID1A. (a) ARID1A expression was analyzed by Western blot in 786-O and ACHN cells treated with genistein (25 μM) or control (DMSO) for 96 hours. (b) 786-O and ACHN cells were transfected with control vector (pcDNA3.1(+)) or MS2-tagged HOTAIR vector and RNA pull-down assays were performed. Pulled-down ARID1A was detected by Western blot. (c) Protein RNA-binding site prediction software, OmiXcore, indicated that ARID1A may directly bind to HOTAIR. High-scoring HOTAIR nucleotides for ARID1A binding are in red, and low-scoring nucleotides are in white. (d) Potential biding sites of ARID1A with HOTAIR. RNA-binding site prediction software (https://sysimm.ifrec.osaka-u.ac.jp/aarna/index.php) shows high-scoring residues in red, and low-scoring residues in blue. (e) ARID1A from 786-O and ACHN cells cultured with genistein (25 µM) or control (DMSO) for 96 hours were pulled down using anti-ARID1A antibody. HOTAIR associated with ARID1A was detected by quantitative RT-PCR. Data from quantitative RT-PCR are presented with respect to IgG that is set to a value of 1. F, Immunoprecipitated ARID1A was analyzed by Western blot. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.