Fig. 4

ADQ binds to the 36G46A sequence of HOTAIR. a, b Molecular docking model of ADQ bound to HOTAIR to predict the specific binding site. c Binding energy analysis revealed that 36G46A resulted in the strongest free energy and binding stability. A mutation in either sequence greatly reduced the binding energy. d EMSA was performed using 5′ biotin-labeled HOTAIR probes to detect the HOTAIR/ADQ binding activity in the wild-type and mutant groups. e HOTAIR full-length (WT), 5′ domain (5′ 300-mer), and 5′ mutation domain (mut 5′ 300-mer) constructs were stably transfected into U87 cells. RIP assays detected the binding efficiency of the HOTAIR fragment in combination with EZH2 proteins in various group. f RNA levels in immunoprecipitants were determined by qRT-PCR and expressed as the relative fold enrichment after subtracting the matched IgG negative control. g ADQ increased the NLK luciferase activity in U87 WT and 5′ domain cells, whereas ADQ had little effect on 5′ domain with 36G46A mutation cells. A similar pattern was observed in MDA-MB-231 cells