Table 2.

LanthaScreen Assay Protocol Table

Step	Parameter	Value	Description
1	Plate cells	14 μL	5,000 HeLa cells
2	Incubation time	Overnight	37°C, 5% CO2
3	Virus	6 μL	BacMam Histone H3 Reagent
4	Control	5 μL	DMSO
5	Library compounds	5 μL	DZNep, RBC124
6	Incubation time	3 days	37°C, 5% CO2
7	Lysis buffer+antibody	5 μL	6× complete lysis buffer with 3× protease inhibitor, 12 nM antibody
8	Incubation time	2–3 h	Ambient temperature
9	Assay readout	340 and 520, 492 nm	EnVision plate reader, TR-FRET mode

Step Notes

1. 384-well white opaque CulturPlates.

2,6. Plates were sealed with sterile breathable tape.

3. From the BacMam Histone H3K27me3 Cellular Assay Kit.

5. 33 μM to 5 nM dilution series.

7. 6× stock concentration lysis buffer, 100× stock concentration protease inhibitor, 3,400 nM stock concentration Terbium-anti-Histone H3K27me3 antibody.

8. Plates lidded until read, in the dark.

9. Excitation with laser (340 nm), emission filter (520/25, 492/8 nm).

TR-FRET, time-resolved fluorescence resonance energy transfer.