Cells proliferation and invasion assays

Cell proliferation assays were performed using Cell Counting Kit-8 (CCK-8, Dojindo, Kumamoto, Japan) and EdU (RiboBio, Guangzhou, China) reagents. CCK-8 experiments were carried out for consecutive five days by adding 30 μl of CCK-8 reagent with 300μl PBS to each well, and incubated at 37°C for 2h. Viable cells were evaluated by absorbance measurements at 450nm. EdU experiments were carried out for analyzing viability of PC-3 or BPH-1 cells after co-culture with stromal cells for 72h using Cell-Light EdU Apollo 488 In Vitro Imaging Kit (RiboBio) according to the manufacturer's instructions. Cell invasiveness were analyzed by Matrigel (Corning) invasion experiments. 1×10⁵ PC-3 or BPH-1 cells were plated in the upper compartment of Matrigel coated 6.5mm Transwell (8μm pore) with serum free medium. After culture for 48h, the cells in the upper compartment were removed with a cotton swab, and the trans-membrane cells were fixed in 4% paraformaldehyde and stained with crystal violet. Stained cells were counted by photographing five fields/membrane at 100× magnification. All assays were performed in triplicate.