Day 1: Transfection

RPE1 cells were reverse transfected with siRNA (Dharmacon) using Lullaby transfection reagent in 384 well plates.

Day 3: Treatment with 3µM ICRF193, 1µM Nocodazole

Media containing ICRF and Nocodazole compounds were added on top of the culture media.

Day 4: Cell fixation

Plates were fixed with Ethanol/Acidic (9.5:0.5) and stored at -20C.

Day 5: Staining

Cells were stained with MPM2-Cy5 and Pacific Green antibodies.

Readout

Plates are read on ArrayScan microscope and the following measurements are taken:

• number of cells (valid object count - Ch1)
• Av and total intensity of MPM2 per cell (summarised as a mean for each well and % above a fixed threshold) (Ch2)
• other associated data e.g nuc morphology data (Ch1)

Plates read on Acumen Explorer and following measurements taken:

• total cell number per well
• total intensity of MPM2
• %8N and %G1 cells