Ectopic MECP2 expression allows androgen-independent growth of human prostate cancer cells. LNCaP cells were infected with the retrovirus pWB3 or pWB3/MECP2, selected and maintained with blasticidin. (a) Cell extracts were prepared and tested for MECP2 expression by Western blotting. (b) Immunofluorescence analysis was performed to detect MECP2. (c) Growth curve assays. Cells were split and treated every 2 days with or without the AR antagonist. Every week they were counted and seeded into fresh medium. The number of PDs was calculated. Statistics were performed using VisualStat and *means a significant difference with P<0.05. (d) Colony formation assays. Cells were seeded at low density and treated every 2 days with AR inhibitor. After 12 days, cells were fixed and stained with Crystal Violet. Representative dishes are shown. (e) DNA content analysis. Cells were treated as described in (d) and processed for DNA content analysis after 10 days. (f) Soft agar assays. Cells were plated in soft agar medium with or without the AR antagonist. AR antagonist treatment was renewed every 3 days. The number of foci was counted after 3 weeks.