Figure 1. Identification of compounds that induce caspase-3 activation in TNFdependent manner. (A) Schematic of the strategy employed for screening compounds from ChemBridge DIVERSetTM library. HT29 colon cancer cells were treated with the test compounds for 18 h in the presence or absence of TNF (50 ng/ml). Cell lysates were prepared and tested for caspase-3 activity using the DEVD-AMC fluorogenic substrate. (B) The compound screen identified six compounds that induced caspase-3 activation only in the presence of TNF using a DEVDase assay. As a secondary screen compounds were tested by immunostaining cells using an antibody specific for cleaved/active caspase-3. Images were captured and the fraction of cells staining positively for caspase-3 was quantified. AK3 and AK10, two of the most active compounds, were selected for further investigation.