Celltiter glo

The indicated cell lines were seeded in 96-well (3×10³ cells/well) plates and cultured in 100 ul medium containing 10% serum. After 24 hours, cells were treated with or without various concentrations of compounds in 50 μl medium for 24 to 48 hours and the cell viability was measured using the Cell Titer Glo (Promega) according to the manufacturer’s instructions. The cell viability assay for the human prostate organoid MSK-PCaX cells were performed as described previously ^{35 (link), 39 (link)}. For 2-D culture, 5,000 organoids cells per well of a collagen coated 96-well cell culture were plated in 100 ul complete human media with vehicle (DMSO) control or JQ1 (10–3000 nM). Viable cells were counted using CellTiter-Glo (Promega) Luminescent Cell Viability Assay after 72 hours treatment. All cell viability experiments were conducted in triplicate. For 3-D culture, 5,000 organoids cells in 10 ul Matrigel per well were plated in 96-well cell culture plate. Viable cells under 3-day-treatment of 100 ul complete human media with vehicle (DMSO) control or JQ1 (10–3000 nM) were counted using CellTiter-Glo (Promega) Luminescent Cell Viability Assay. Data was shown as mean ± SD from three independent experiments.

ID8-LUC and ID8-VEGF cells were plated at 1,000 cells per well and treated with ampicillin, vancomycin, metrinodazole, and neomycin in the same proportions used in the murine studies. After 72 hours, proliferation was measured using CellTiter-Glo (Promega) and compared to vehicle-treated control. Additionally, following incubation for 7 days with ampicillin, vancomycin, metrinodazole, and neomycin, the IC₅₀ of cisplatin (Spectrum Chemical) was assessed compared to vehicle pretreated controls utilizing CellTiter-Glo (Promega).

Cells were transfected with plasmid or siRNA 24h before being plated for colony formation or CellTiter-Glo assays (Li et al., 2020 (link)). To assay clonogenic survival, cells were seeded at 500–1000 cells/well in 6-well plates in triplicates. Drugs at the shown doses were added after 12 h and cells were permitted to grow for 14 days. Colony formation was scored by fixing and staining with 0.5% (w/v) crystal violet in 20% methanol. For short term CellTiter-Glo survival assays, cells were plated in 96-well plates at 800–1000 cells/well, and treated with drugs at the indicated concentrations after 12 h. Three days later, cellular viability was measured using CellTiter-Glo (Promega). Survival at each drug concentration was calculated as a percentage normalized to the corresponding untreated control, for both assays.

Cells were seeded in a 96-well plate and incubated for 12–24 h prior to initial treatment. The number of cells per well was chosen empirically based on cell growth rates (LNCaP, 5000 cells/well; PC3 and RWPE-1, 3000 cells per well; HEK293T, 1000 cells/well). Cells were treated with either DMSO control or serial dilutions of inhibitors, and media with compound was refreshed every 48 h. Cells were harvested when control samples reached confluence (293T cells, 5 days; PC3 cells, 6 days; and RWPE-1 and LNCaP cells, 7 days), and cell viability was measured via CellTiter-Glo (Promega).
Knockdown with compound treatment: LNCaP cells were transfected with either PBRM1 knockdown or vector control and treated for 48 h with (1:1000) puromycin. Cells (5000) were seeded in a 96-well plate for 12 h. Cells were treated with 16 or DMSO control for 5 days, with media changes every 48 h, and cell viability was measured via CellTiter-Glo (Promega).