Cell Viability Assay. MV4;11 cells were incubated with compounds at the desired concentration for 48h on a clear-bottom 384-well plate. MV4;11 cells were kept in RPMI medium supplemented with 10% FBS, L-glutamine. Initial cell density was 3 × 10 5 per mL. The cells were treated with various concentrations of compound or 0.05% DMSO in triplicates for each concentration point. After treatment, cell viability was measured with Promega CellTiter-Glo luminescent cell viability assay kit according to the manufacturer instructions. Signal was recorded on a BMG Labtech PHERAstar luminescence plate reader with recommended settings. Data were analyzed with GraphPad Prism software to obtain EC50 values of each test compound. Kinetic Degradation, Quantitation, and compound washout experiments. HEK293 cells (ATCC) stably expressing LgBiT (Promega) were edited using CRISPR/Cas9 to endogenously HiBiT tag the Nterminal genomic loci of BRD2, BRD3, or BRD4 . For kinetic degradation assays, cells were plated in quadruplicate in white 96-well tissue culture plates at a density of 2 × 10 4 cells per well in 100µL of growth medium and incubated overnight at 37°C, 5% CO2. The following day, medium was replaced with CO2-independent medium (Gibco) containing a 1:100 dilution of Endurazine (Promega) and were incubated at 37°C in 5% CO2 for 2.5h before addition of a 3-fold serial dilution of the indicated concentrations of SIM1, SIM2, SIM3, or ARV-771 (MedChemExpress). Plates retaining lids were placed into the GloMax Discover Microplate Reader (Promega) set to 37°C, and continuous luminescent measurements with readings every 5-15min were made over a 22-24h period. Degradation rate (λ), degradation rate plateau (λmax), and degradation plateau (Dmax) were calculated from above determined kinetic degradation profiles. Briefly, the degradation portion of each kinetic concentration curve was fitted to a single exponential equation where ƛ = degradation rate in units of h -1 . The degraded fraction, Dmax, was calculated as 1plateau. For each curve, the data points before onset of degradation were excluded from the fits. The Dmax was then plotted against concentration to determine Dmax50 values. For compound washout assays, the CRISPR HiBiT BRD2, BRD3, and BRD4 HEK293 cells were plated as described above for kinetic assays and treated with SIM1 (100nM and 10nM), (R,S)-SIM1 and MZ1 (both at 100nM), and equivalent volume of DMSO for a period of 3.5h, with continual luminescence monitoring. The concentrations were chosen to allow achieving >70% Dmax of all three BET proteins at 3.5h with all three compounds, with the caveat that they reflect different relative ratios with DC50 values for each protein-compound combination. At 3.5h, media was removed and replaced with CO2-independent medium containing Endurazine. The plates were placed back in the luminometer for continued monitoring of protein levels for a further 46.5h.