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. 2021 Aug 11;12:4860. doi: 10.1038/s41467-021-24859-2

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© The Author(s) 2021

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 4 — a Scheme of the GPX4 arm of ferroptosis. b Cell viability dose response curve at 24 h, n = 3 biological replicates. c EC₅₀ fold changes in dose response following GOT1 knockdown and RSL3 treatment, n = 3 biological replicates, ****P ≤ 0.0001, **P = 0.005128, **P = 0.003314, **P = 0.008921, ***P = 0.000691. d % Cytotoxicity following GOT1 knockdown and RSL3 treatment at 24 h. Cytotoxicity was measured by LDH (Lactate dehydrogenase) release and normalized to a cell lysis control, n = 3 biological replicates, n.s. P = 0.1763, ****P = < 0.0001, ****P < 0.0001, ****P < 0.0001. e Relative lipid ROS in Pa-Tu-8902 iDox-shGOT1 treated with 32 nM RSL3 or 750 nM Erastin −/+ 1 μM Ferrostatin-1(Fer-1) for 6 h (n = 3 biological replicates), **P = 0.0057, ****P < 0.0001,****P < 0.0001,****P < 0.0001. f Cell viability of Pa-Tu-8902 iDox-shGOT1 cultured in vehicle (0.1% DMSO) −/+ dox (black and light gray), drug (32 nM RSL3, 750 nM Erastin, 40 μM BSO) −/+ dox (gray and red), or drug and dox (blue) in the presence of lipophilic antioxidants 1 μM Fer-1 and 100 μM Trolox, or an iron chelator 10 μM DFO (deferoxamine), ****P ≤ 0.0001 dox and dox/RSL3, Erastin, BSO; ****P < 0.0001 dox/RSL3 and dox/RSL3, Erastin, BSO/Fer-1, Trolox, DFO. Viability was assessed after 24 h of treatment for RSL3 and Erastin conditions and 72 h for BSO treatment conditions. GOT1 was knocked down for 5 days prior to treatment. Data are normalized to the –dox and vehicle-treated control (n = 3 biological replicates). g Cell viability following the procedure in (f) but in the presence of 10 μM Necrostatin-1 (Nec-s, necroptosis inhibitor), 50 μM ZVAD-FMK (Z-Vad, apoptosis inhibitor), or 1 nM bafilomycin A1 (BA-1, lysosomal acidification inhibitor), n = 3 biological replicates, ****P ≤ 0.0001 dox and dox/RSL3, Erastin, BSO; dox/Erastin, dox/Erastin/Nec-s, ****P ≤ 0.0001; dox/Erastin, dox/Erastin/Baf-A1, ***P = 0.0002. dox/BSO, dox/BSO/Nec-s, ***P = 0.0021. Error bars represent mean ± SD. Two-tailed unpaired T-test or one-way ANOVA: Non-significant P > 0.05 (n.s., as noted). Source data are provided as a Source Data file. GPX4 glutathione peroxidase 4.