Estrogen receptor β (ERβ) is required for apoptosis induction by TSN. (A) U87 cells were treated with 10 nM TSN either with or without the selective ERβ antagonist PHTPP for 48 h and evaluated for Bcl-2, Bax, phosphorylated Akt, and p53 expression; (B) U87 cells were transfected with a specific siRNA against ERβ and treated with 10 nM TSN for an additional 48 h. The expression of Bcl-2, Bax, pAkt, and p53 were analyzed by Western blot. siRNA against GFP was used as a control for the transfection procedure; (C) Flow cytometry analysis of apoptosis in U87 cells transfected with siERβ either with or without TSN treatment. siRNA targeting GFP was used as a control; (D) ERβ-negative T98G cells were transfected with either an ERβ-expressing plasmid or empty vector (EV), treated with 10 nM TSN, and evaluated for Bcl-2, Bax, and pAkt protein levels; (E) The T98G/EV and T98G/ERβ cells were treated with either TSN or vehicle for 48 h and evaluated for apoptosis by flow cytometry.