PC-3-4H7 cell is both Steap1and PSMA antigen high express cells. To run the assay,the cells (180 ul,6000cells)were added to each well in a 96-well plate and incubated for 24 hours at 37°C with 5%CO ₂. Next,the cells were treated with test compounds (20ul)at various concentrations in appropriate cell culture medium (total volume,0.2 mL).The control wells contain cells and the medium but lack the test compounds.The plates were incubated for 120 hours at 37°C with 5%CO ₂.MTT (5 mg/mL)was then added to the wells (20ul)and the plates were incubated for 1.5 hr at 37°C.The medium was carefully removed and DMSO (180ul)was added afterward.After it was shaken for 15min, the absorbance was measured at 490 nm and 570 nm with a reference filter of 620 nm.The inhibition% was calculated according to the following equation:inhibition%=[1- (assay-blank)/ (control-blank)]× 100.

C4-2B is both Steap1 and PSMA antigen high express cells.To run the assay,the cells (180 ul,6000cells)were added to each well in a 96-well plate and incubated for 24 hours at 37°C with 5%CO ₂. Next,the cells were treated with test compounds (20ul)at various concentrations in appropriate cell culture medium (total volume,0.2 mL).The control wells contain cells and the medium but lack the test compounds.The plates were incubated for 120 hours at 37°C with 5%CO ₂.MTT (5 mg/mL)was then added to the wells (20ul)and the plates were incubated for 1.5 hr at 37°C.The medium was carefully removed and DMSO (180ul)was added afterward.After it was shaken for 15min, the absorbance was measured at 490 nm and 570 nm with a reference filter of 620 nm.The inhibition% was calculated according to the following equation:inhibition%=[1- (assay-blank)/ (control-blank)]× 100.

22RV1 is both Steapl and PSMA antigen low express cells.To run the assay,the cells (180 ul,6000cells)were added to each well in a 96-well plate and incubated for 24 hours at 37°C with 5%CO ₂. Next,the cells were treated with test compounds (20ul)at various concentrations in appropriate cell culture medium (total volume,0.2 mL).The control wells contain cells and the medium but lack the test compounds.The plates were incubated for 120 hours at 37°C with 5%CO ₂.MTT (5 mg/mL)was then added to the wells (20ul)and the plates were incubated for 1.5 hr at 37°C.The medium was carefully removed and DMSO (180ul)was added afterward.After it was shaken for 15min, the absorbance was measured at 490 nm and 570 nm with a reference filter of 620 nm.The inhibition% was calculated according to the following equation:inhibition%=[1- (assay-blank)/ (control-blank)]× 100.