Exogenous Monounsaturated Fatty Acids Promote a Ferroptosis-Resistant Cell State

This experiment tested the hypothesis that an exogenous MUFA, in this case oleic acid (125 µM), would suppress cell death in response to different lethal stimuli in HT-1080^N cells. Experimental details are as follows:

A library of 261 bioactive compounds (Cat# L2000) was obtained from Selleck Chemicals (Houston, TX) and stored at -80°C. The library was re-formatted from 96-well to 384-well format using a Versette automated liquid handler configured with a 96-channel pipetting head, and diluted to 2 mM in DMSO. The day before screen, 1,500 HT-1080N cells/well were seeded into two 384-well plates in 40 μL medium. The next day, the medium was removed and replaced with medium containing SYTOX Green (20 nM final) and compounds from a freshly thawed library master stock plate (1 compound/well) were added to a final concentration of 5 μM, using a Versette liquid handler equipped with a 384-channel pipetting head. One plate was co-treated with EtOH and the other with oleic acid (125 µM). Plates were imaged immediately and every 4 h thereafter for a total of 120 h using the Essen IncuCyte Zoom. Counts of SYTOX Green and mKate2 objects per mm2 were obtained and the lethal fraction calculated as described in (Forcina et al., 2017). The area under the curve (AUC) of lethal fraction scores across the full 120 h were calculated using the trapezoid rule in Prism 7.0.