Fig. 2

ERα binds directly to the lncRNA NCALD promoter in BC cells, thereby regulating the expression of lncRNA NCALD. Ability of ERα to bind lncRNA NCALD. The DNA–protein complexes were separated through electrophoresis and identified as 'shifts' from the position of the unbound probe. The inclusion of an additional goat anti-ERα antibody resulted in a noticeable 'supershift' and perturbed the 'shift' (panel a). A ChIP assay was performed to investigate the binding of ERα to the promoters of lncRNA NCALD in MCF7 and T47D cells using shRNA targeting ERα or control shRNA (*P < 0.05) (panel b). All experiments were performed in triplicate. RT-qPCR assay showing the expression of ERα and lncRNA NCALD (panel c). All experiments were performed in triplicate. The plasmid details were used in a dual-luciferase reporter assay (panel d). The orange box indicates the location of the mutation site for ERα binding, whereas the arrow denotes the transcription start site. Plasmids containing the wild-type lncRNA NCALD promoter and those with a mutated lncRNA NCALD promoter were transfected into shRNA ERα and control cells. All experiments were performed in triplicate. (*P < 0.05, **P < 0.01, ***P < 0.001)