Whole genome pooled shRNA screen to characterize Raptinal-induced apoptosis in the MIA PaCa-2 cell line. Homo sapiens

The goal of the study was to characterize the mechanism by which Raptinal induces apoptosis using a genome-wide pooled shRNA screen. Following drug treatment, the abundance of the shRNA constructs were determined and compared between DMSO and Raptinal-treated samples. Constructs depleted in Raptinal samples versus DMSO control, are expected to potentiate Raptinal-induced apoptosis by down regulating anti-apoptotic proteins. In contrast, shRNA constructs enriched upon Raptinal treatment are expected to target pro-apoptotic proteins, whose down regulation confers protection from Raptinal-induced apoptosis. Follow up experiments were performed on enriched shRNAs in order to validate potentially novel pro-apoptotic proteins. Overall design: Cells were treated with Raptinal (1.25-1.8 micromolar) or DMSO vehicle control over a 20 day period. After every 2-3 days of drug treatment, cells were allowed to recover in drug-free medium and treatment repeated for a total of 3 rounds over the 20 day period. Two biological replicates of Raptinal-treatment were performed and one biological replicate of DMSO vehicle control was performed. Cell passaging was performed on a scale sufficient to maintain >100 fold library representation. At the end of the 20 days, total RNA was extracted and shRNA abundances determined.