Background
Erastin2 is a potent system xc- inhibitor and ferroptosis-inducing agent [1].
Erastin2 (5μM, 24h) significantly inhibited the activity of human HAP1 haploid cells, and that erastin2-induced cell death was inhibited by co-treatment with the lipophilic radical-trapping antioxidant ferrostatin-1 or the iron chelator deferoxamine (DFO), but not the pan-caspase inhibitor Q-VD-OPh [1]. Erastin2 induced ferroptosis in HT-1080(1 μM), T98G glioblastoma (1 μM), and A549 non-small-cell lung carcinoma cells (2 μM), and non-transformed IMR-90 diploid fibroblasts (125nM) [2]. Erastin2 (1 μM) inhibited system xc- function, depleted total glutathione (GSH + GSSG), and upregulated CHAC1 expression [2]. Erastin2 (1-10μM) increased FSP1KO cells sensitivity [3]. In the context of erastin2-induced ferroptosis, the onset of cell death correlates with the oxidation of the lipid peroxide-sensitive probe C11 BODIPY 581/591 (C11) (Cat.No. GC40165) specifically at the plasma membrane, linking the onset of cell death to a key terminal marker of the ferroptotic process [1].
参考文献:
[1]. Cao JY, Poddar A, Magtanong L, Lumb JH, Mileur TR, Reid MA, Dovey CM, Wang J, Locasale JW, Stone E, Cole SP. A genome-wide haploid genetic screen identifies regulators of glutathione abundance and ferroptosis sensitivity. Cell reports. 2019 Feb 5;26(6):1544-56.
[2]. Magtanong L, Ko PJ, To M, Cao JY, Forcina GC, Tarangelo A, Ward CC, Cho K, Patti GJ, Nomura DK, Olzmann JA. Exogenous monounsaturated fatty acids promote a ferroptosis-resistant cell state. Cell chemical biology. 2019 Mar 21;26(3):420-32.
[3]. Bersuker K, Hendricks JM, Li Z, Magtanong L, Ford B, Tang PH, Roberts MA, Tong B, Maimone TJ, Zoncu R, Bassik MC. The CoQ oxidoreductase FSP1 acts parallel to GPX4 to inhibit ferroptosis. Nature. 2019 Nov 28;575(7784):688-92.
Erastin2 是一种有效的系统 xc 抑制剂和铁死亡诱导剂[1]。
Erastin2 (5μM, 24h) 显着抑制人 HAP1 单倍体的活性细胞，并且 erastin2 诱导的细胞死亡被亲脂性自由基捕获抗氧化剂 ferrostatin-1 或铁螯合剂去铁胺 (DFO) 共同治疗抑制，但泛半胱天冬酶抑制剂 Q-VD-OPh [ 1]。 Erastin2 在 HT-1080（1 77777#181；M）、T98G 胶质母细胞瘤（1 77777#181；M）和 A549 非小细胞肺癌细胞（2 77777#8181；M）和未转化的 IMR-90 二倍体成纤维细胞（ 125nM）[2]。 Erastin2 (1 μM) 抑制系统 xc- 功能，耗尽总谷胱甘肽 (GSH + GSSG)，并上调 CHAC1 表达 [2]。 Erastin2 (1-10μM) 增加 FSP1KO 细胞敏感性 [3]。在 erastin2 诱导的铁死亡的背景下，细胞死亡的发生与脂质过氧化物敏感探针 C11 BODIPY 581/591 (C11)（货号 GC40165）的氧化相关，特别是在质膜上，与细胞死亡是铁死亡过程的关键终末标志物[1]。
Protocol
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Cell lines
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Caki-1 cells
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Preparation Method
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Caki-1 cells were seeded in a 96-well dish at 4000 (cells treated with control shRNA constructs and nutlin-3) or 2000 (all other treatments) cells/well and infected with lentiviruses carrying non-targeting shRNAs (sh-NT) or shRNAs targeting CDKN1A at an M.O.I. of ~1 in media containing 8 μg/mL Polybrene (source). Infected cells were then spun at 1000 rpm for 1 h at room temperature. The next day, virus-containing medium was removed, and cells were incubated in medium containing puromycin (10 μg/mL) and DMSO or nutlin-3 (10 μM) for a further 24 h. Media was then removed and replaced with media containing SYTOX Green (0.022 μM), DMSO or nutlin-3 (10 μM), and DMSO or erastin2 (1 μM), and cell death was assayed for the subsequent 48 h using time-lapse imaging. Cell death was assessed by counting SYTOX Green positive cells over time.
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Reaction Conditions
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1 μM, for 48 h
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Applications
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Short hairpin RNA (shRNA)-mediated silencing of CDKN1A in Caki-1 cells likewise abrogated the ability of nutlin-3 pretreatment to delay subsequent erastin2-induced cell death.
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参考文献:
[1]: Dixon SJ, Patel DN, Welsch M, Skouta R, Lee ED, Hayano M, Thomas AG, Gleason CE, Tatonetti NP, Slusher BS, Stockwell BR. Pharmacological inhibition of cystine-glutamate exchange induces endoplasmic reticulum stress and ferroptosis. elife. 2014 May 20;3:e02523.
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