Figure 3

AR is a suppressor in EMT of S/P cells from LNCaP cell line. S/P Cells were infected with lentivirus carrying either vector or AR and grown in two different androgen concentrations, charcoal stripped (CS)-FBS + 1 nM DHT, CS-FBS + 10 nM DHT. (A) Western blot analysis of AR, E Cadherin, N Cadherin, Vimentin and Snail using cell extracts of vector S/P and AR S/P cells. (B) MTT assay. Cells were incubated in 24 well dishes (1 × 10⁴ cells/well). At 0, 2, 4, and 6 days, MTT assays were performed. (C) Soft agar colony formation assay. The plates were incubated at 37°C in a humidified incubator for 21 days. Colonies larger than 1 mm in diameter were counted. (D) Sphere formation assay. Cells (5,000) were mixed with Matrigel (1:1 ratio, total 200 μl) and placed in rim of well of 24well culture plates. After solidified, 1 ml of medium was added and grown for 21 days. Pictures were taken under microscope. (E) Migration assay. The migrative potential of different cells was assessed using the 8 μm pore Transwell system. Cells (1 × 10⁵) were resuspended in 100 μl serum free medium and added in the upper compartment. 600 μl of 20% CS-FBS medium was placed in the lower compartment. After 24 hours, cells that had not migrated were removed from the upper face of the membranes with cotton swabs. The average number of cells per field of view (five random fields per membrane) was counted at × 20 magnification. All experiments samples were plated in triplicate.