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.

本实验旨在验证外源性单不饱和脂肪酸（MUFA），在本例中为油酸（125 µM），能够抑制HT-1080^N细胞对多种致命刺激产生的细胞死亡假说。实验细节如下：从赛莱克化学公司（休斯顿，德克萨斯州）获取了包含261种生物活性化合物的库（产品编号L2000），并将其储存在-80°C的条件下。该库通过配备96通道移液头的Versette自动液体处理器从96孔板格式转换为384孔板格式，并稀释至2 mM的DMSO溶液中。在筛选前一天，将1,500个HT-1080N细胞/孔接种于两个384孔板中，加入40 μL的培养基。次日，移除培养基并替换为含有SYTOX Green（终浓度为20 nM）的培养基，并使用配备384通道移液头的Versette液体处理器，从新鲜解冻的库主储备板（每孔1种化合物）添加化合物至终浓度为5 μM。一个板与乙醇共处理，另一个板与油酸（125 µM）共处理。使用Essen IncuCyte Zoom在加入化合物后立即以及之后的每4小时进行一次成像，总共持续120小时。通过计数每平方毫米的SYTOX Green和mKate2对象的数量，计算出致死分数，具体方法见(Forcina等人，2017年)。使用Prism 7.0中的梯形规则计算了120小时内致死分数得分曲线下的面积（AUC）。