MECP2 maintains the c-myc level to allow androgen-independent growth. (a) LNCaP cells stably infected with the pWB3 (control) or pWB3/MECP2 retroviral vector were treated with AR antagonist for 0, 5, or 10 days. Cell extracts were prepared and analysed for MECP2, c-myc and ODC. (b) PC-3 cells were infected with pRS or pRS/MEP2 vectors and puromycin selected. After 4 days, cell extracts were prepared and levels of c-myc protein were analysed by Western blotting. (c) Cells stably expressing MECP2 or c-myc or infected with the control vector were cultured for 12 days in the presence of the AR antagonist. Cells were fixed and stained with Crystal Violet. Representative dishes are shown. (d) RNA from control or MECP2-expressing cells was extracted and analysed by RT–PCR for expression of c-myc. Actin is used as a loading control. (e) LNCaP cells with or without MECP2 were treated with 2 μg/ml of actinomycin D during the indicated time. RNA was prepared and analysed by RT–PCR for c-myc expression. (f) Control or MECP-2 LNCaP-expressing cells were treated with 10 μg/ml of cycloheximide during the indicated time. Cell extracts were prepared and analysed for level of c-myc protein. (g) PC-3 cells infected with pRS or pRS/MECP2 vectors were treated with cycloheximide during various times. C-myc protein level was analysed by immunoblotting.