Fig. 1.

Measurement of H3K27me3 level in SU-DHL-6 cells using AlphaLISA format. (A) Titration of SU-DHL-6 cells in H3K27me3 AlphaLISA assay. The bar graph illustrates the responses of the negative (uninhibited) controls (▪) versus positive (100% inhibited) controls (□) at various cell densities (0–15,000 cells per well). (B) Response of AlphaLISA assay at various peptide concentrations. Five thousand SU-DHL-6 cells were seeded to assay plates. Serially diluted peptides from 30 pM to 3 μM were added to the assay plate just before the addition of the AlphaLISA detection reagents. (C) The concentration response of reference inhibitors in H3K27me3 AlphaLISA cell-based assay. Cells were incubated with a compound for 3 days at 37°C in 5% CO₂. Evaluation of the cellular inhibitory activity of the reference inhibitor generated average IC₅₀ values of 13.0±1.3 and 1.4±0.4 μM for RBC124 (▪) and DZNep (▾), respectively. The average±SEM IC₅₀ value from three independent determinations was calculated. Error bars denote SEM. H3K27me3, trimethyl histone H3 Lysine 27; DZNep, 3-deazaneplanocin A; IC₅₀, half-maximal inhibitory concentration; SEM, standard error of the mean.