Fig. 2.

Measurement of H3K27me3 level in HeLa cells using LanthaScreen technology. (A) Optimization of BacMam histone H3 transduction versus HeLa cell plating density in H3K27me3 LanthaScreen cell-based assay. Cells were plated for 5,000, 10,000, and 15,000 cells per well. The bar graph illustrates the assay windows of 10% (□) and 30% (▪) BacMam histone H3 virus transduction, followed by incubation with an internal control compound (100% inhibition, positive control) and DMSO (0% inhibition, negative control) for 3 days at 37°C in 5% CO₂. The assay window was calculated as the emission ratio of 520 and 490 nm of the negative control/emission ratio of 520 and 490 nm of positive control. (B) The concentration response of reference inhibitors in H3K27me3 LanthaScreen cell-based assay. Cells were transduced with 30% BacMam histone H3 virus, followed by treatment of compounds RBC124 (▪) and DZNep (▾) for 3 days. Evaluation of the cellular inhibitory activity of the reference inhibitors generated average IC₅₀ values of 20.3±3.5 μM and 21.0±6.9 nM for RBC124 (▪) and DZNep (▾), respectively. The IC₅₀ value (average±SEM) from three independent determinations was calculated. Error bars denote SEM. DMSO, dimethylsulfoxide.