PF-06726304

Karpas-422 cells are plated in complete cell culture medium (100 μL/well) in a 96-well clear, V bottom polystyrene cell culture plate at a density of 2500 cells/well. The cells are then incubated for 2−3 h at 37 °C and 5% CO2. Compound dilution plates are prepared in 96-well clear, U-bottom polypropylene plates in 100% DMSO at 10 mM stock concentration in duplicate wells using an 11-point serial dilution (1:3 dilutions). This compound is further diluted in growth medium, and 25 μL is added to each well of the cell plates. The highest this chemical concentration tested is 50 μM final, with a 0.5% final DMSO concentration. The plates are then incubated for 72 h at 37 °C and 5% CO2. At the end of the incubation period with this compound, the plates are centrifuged at 2000 rpm for 5 min at room temperature, and the medium is removed. Then 100 μL of acid-extracted solution is added to each well. The plates are shaken for 50 min at 4 °C to lyse the cells, and then 38 μL of neutralization buffer is added. The plates are then frozen at −80 °C. The next day, the plates are shaken at room temperature to thaw. Cell lysates (50 μL/well) are then transferred to ELISA plates. The plates are covered and incubated for 2.5 h at room temperature with constant slow-speed shaking. After incubation, the plates are washed seven times (300 μL/well) with 1× wash buffer. Then 100 μL of biotinylated trimethylhistone H3K27 detection antibody is added to each well and incubated for 2 h at room temperature with constant slow-speed shaking. After incubation, the plates are washed as described above. Horseradish peroxidase-linked antibody (100 μL) is added to each well and incubated for 60 min at room temperature with constant slow-speed shaking. After incubation, the plates are washed. Finally, 100 μL of TMB substrate reagent is added to each well and incubated for 5 min at room temperature in the dark with slow shaking, after which 100 μL of stop solution is added to stop the reaction.