Cell proliferation was evaluated using the tetrazolium compound WST-8 ([2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl))-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt) according to the manufacturer’s instructions (Cell Counting Kit-8 [CCK-8]; Dojindo Laboratories, Kumamoto, Japan). At the time of the assay, cells cultured for 4 to 5 days were harvested by trypsinization, and cell viability was evaluated by trypan blue exclusion. Cells were quantified (Coulter counter; Beckman-Coulter, Fullerton, CA) and cultured in 96-well plates: each well contained 104 cells in a total volume of 100 μL. Each assay included one plate. The plate included blank wells (0 cells/0.1 mL), control wells (104 cells /0.1 mL), control wells treated with L-733,060, and control wells treated with different concentrations of SP (with or without L-733,060). The plates were inoculated with L-733,060 (7.5, 10, 15, and 20 μM for WERI-RB-1 and 10, 15, 20, and 25 μM for Y-79) and were incubated for 49 and 40 hours, respectively. The plates were also inoculated with SP (10, 50, and 100 nM), with (10 μM: WERI-RB-1 and 15 μM: Y-79) and without L-733,060 for their first doubling times (49 or 40 hours). For the proliferation assays, 10 μL of the CCK-8 reagent was added to each well 90 minutes before reading the samples on a multiscanner microplate reader (Multiskan Spectrum, Thermo Labsystems, Barcelona, Spain) at 450 nm. The quantity of product, as measured by optical density, is directly proportional to the number of living cells. Each experimental condition (blank wells, control wells, and control wells treated with different concentrations of L-733,060 or SP) was assayed in duplicate, and all experiments were performed at least three times. The IC50 of L-733,060 was calculated with a curve-fitting parameter.