Facs express software

For live-cell FACS analysis, cells were harvested, washed once in phosphate-buffered saline (PBS), and incubated with antibodies in 1× PBS for 1 hr on ice. Ghost Dye Violet 510 was used to discriminate live cells. For FACS analysis of fixed cells, cells were processed as described previously (Diab et al., 2020 (link)), with the following modifications. For DNA replication analysis, cells were pulsed with 10 μM 5-ethynyl-20-deoxyuridine (EdU) for 20–60 min. Following EdU labeling, cells were harvested using TrypLE Express (Thermo Fisher Scientific) and fixed in 2% paraformaldehyde (PFA) for 20 min at room temperature, washed once with PBS, resuspended in ice-cold methanol, and allowed to sit overnight at –20°C prior to further processing. Cells were then washed twice with 1 ml BD Perm/Wash Buffer (BD Biosciences), Following a Click-iT step for EdU labeling according to manufacturer’s protocol (Thermo Fisher Scientific), cells were incubated with the appropriate primary antibodies in 3% BSA in PBS for 1 hr at room temperature. Cells were washed, incubated in secondary antibody for at least 1 hr, and in DAPI (1:1000, BD Biosciences, 564907) for at least 15 min, washed again, and resuspended in Stain Buffer (FBS—BD Biosciences). Samples were run on a BD FACSCelesta or FACSymphony, visualized with FACS Express software (DeNovo), and analyzed using FlowJo software v10.6.1.

Cells were processed and DNA content analysed using flow cytometry as described previously [68 (link)]. The percentage of cells in each cell cycle phase was calculated with ModFit LT (Verity Software House) based on DNA histograms of 20,000 cells per treatment. For measuring GFP expression in LNCaP–iGFP cells treated with or without Dox, cells were harvested and immediately analyzed using a FC500 flow cytometer (Beckman Coulter) and data analyzed using FACS Express software (DE Novo Software).