External ID: CBX7-Alpha-primary

Protocol: The protocol for the CBX7 AlphaScreen that we use is as follows: We create a standard alpha buffer (50mM HEPES, 150mM NaCl, 0.1% w/v BSA, 0.01% w/v Tween20) at pH 8.0 and make up two stock solutions at 2x final concentration in the alpha buffer. Solution A contains Human His6-CBX7 is used with biotinylated-H3K27me3 (residues 20-34), at 100nM and 50nM respectively. Solution B contains 20nM streptavidin donor beads and 20nM nickel acceptor beads. For 384 well assay formats, 10uL of solution A is added to each well of the assay plate and the plate is spun at 1000 rpm for 30 seconds. 100 nL of experimental ccompounds from stock plates are delivered by robotic pin transfer using a Janus Workstation (PerkinElmer), allowing the compounds to interact with CBX7 binding prior to assay measurement, followed by another spin and an incubation room temperature. Finally, 10uL of solution B is added to each well, the plate is spun again and then incubated at room temperature. AlphaScreen measurements are performed on an Envision 2104 (PerkinElmer) utilizing the manufacturer's protocol that has the correct excitation and emission wavelengths, cutoff filters, delay time, etc. We also do a crosstalk calculation through the Envision software to correct for luminescence for adjacent wells while reading the plate. The signal is then normalized to DMSO control wells on the compound plate prior to creation of the IC50 curves.

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External ID: TELO_01

Protocol: 50000 A2780 cells were seeded in wells white 96 well plates (Greiner) in complete RPMI medium (Life Technologies) for 24h prior to transfection. Each well was transiently transfected with 250ng of reporter plasmid using Superfect reagent (Qiagen) at 2.5:1 ratio of reagent (ul): plasmid (ug), according to the manufacturer's protocol. Transfection complexes were allowed to form for 15 minutes prior to addition to cells. Cells were incubated with the transfection complex for 2.5h then rinsed in PBS and returned to fresh growth medium. 32h post transfection, compounds dissolved in DMSO and diluted in growth medium were added in triplicate at a final assay concentration of 20uM. In each plate, 12 wells were treated with DMSO only. Cells were incubated with compounds for a further 16h prior to harvest. Cells were then rinsed once in PBS, lysed for 15 minutes in 30ul Passive Lysis Buffer per well (Promega) with shaking on a vortex with plate attachment. Luciferase activity was detected using 50ul per well of freshly reconstituted Luciferase Assay Reagent (Promega). Plates were read on a GloMax-96 microplate luminometer. Luciferase activity of compound-treated wells was compared with the average of the DMSO controls to generate fold-change readout.

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