Cell proliferation assay, IC50 assay, and drug response assay

For growth curve of NIH3T3 and RWPE-1 cell lines, indicated cells were seeded at 3000 cells per well in a 96-well plate in quadruplicate, treating with increasing drug concentrations for 48 hr. Foretinib (#A2974) was purchased from ApexBio. MK2206 (#HY-10358) was purchased from MCE. The viability rate of each cell line treated with different drug concentrations was normalized to that of the corresponding untreated cells. The in vitro half-maximal inhibitory concentration (IC₅₀) values were determined using Orange 8.0 software and dose–response curves were plotted with package drc in R environment. For proliferation assay of RWPE-1, PC3, or LNCaP cells, indicated cells were seeded at 3000 cells per well in a 96-well plate in quadruplicate for 6 d (RWPE-1), 8 d (PC3), and 9 d (LNCaP), respectively. The viability of cells was determined using a Celltiter-Glo assay (Promega) on INFINITE 200 Pro multimode reader (TECAN). For drug response assay, NIH3T3 cells stably expressing the FOXP2 and FOXP2-CPED1, respectively were suspended in 0.4% soft agar at 3000 cells (cell suspension in complete growth medium mixed with 2× DMEM, 20% FBS, and 1.2% agar at a 1:1:1 dilution) with indicated drug concentrations. Each cell line was plated in quadruplicate. The medium with indicated drug concentration was changed every 4 d and incubated for 4 wk. The colony counting was performed using a Nikon microscope and representative images were then acquired. RWPE-1 cells stably expressing the FOXP2 and FOXP2-CPED1, respectively, were plated at a density of 3000 cells per well in triplicate in a 6-well plate for 12 d with indicated drug concentrations, then fixed with 4% paraformaldehyde solution for 10 min and subsequently stained with 0.05% crystal violet.