Fig. 3

ADQ blocks the HOTAIR/EZH2 interaction and inhibits the H3K27-mediated tri-methylation of NLK. a RIP assays were performed to detect the binding efficiency of the HOTAIR fragment in combination with EZH2 proteins. b RNA levels in immunoprecipitants were determined by qRT-PCR and expressed as the relative fold enrichment after subtracting the matched IgG negative control. c, d ChIRP was used to assess the binding activity of HOTAIR with EZH2 and the NLK promoter in control and ADQ-treated U87 and MDA-MB-231 cells. e NLK and H3K27me3 protein levels were detected by Western blotting. f ChIP was performed with anti-H3K27me3 and anti-H3K27Ac antibodies in cells treated with DMSO or ADQ, and anti-IgG served as the negative control. Chromatin obtained by pull-down was used for qPCR to amplify the promoter region of NLK located 2 kb upstream of the transcription start site. g Fold enrichment of H3K27me3 and H3K27Ac on the NLK promoter normalized to the IgG signal after normalization to the respective input signal