CCT007093 is an effective PPM1D (WIP1) inhibitor (IC50: 8.4 μM).

PPM1D:8.4 μM

In vitro phosphatase assay: Recombinant PPM1D (20-50?pmol) is diluted in Tris buffer (50?mM, pH 8), NaCl (100?mM), β-mercaptoethanol (1?mM) or DTT (1?mM) and treated with MnCl2 (0, 1, 10 and 20?mM) or MgCl2 (0 and 40?mM). Where appropriate, inhibitors of PPM1D (10-50?μM) are added and the assay mix incubated for 30?min at room temperature. Recombinant phospho-P38 (200?pmol) is then added and the mixture incubated at 37°C for 1?h. The reaction is quenched by the addition of excess ethylenediaminetetraacetic acid (EDTA), sodium dodecyl sulphate-sample loading buffer and boiling for 5?min at 95°C followed by gel electrophoresis and western blotting.

Cells are transfected with a pSUPER plasmid and an additional plasmid expressing the blasticidin resistance gene (pEFBsd) in a molar ratio of 10:1. Cells are plated in 10?cm plates 24?h after transfection. Blasticidin selection (5?μg/ml) is initiated 48?h post-transfection and replenished every 3 days. Colonies are fixed in methanol and stained with crystal violet after 14 days. Colonies are quantified on a Colcount and the surviving fraction (SF) determined.(Only for Reference)