p53 does not significantly impact the induction of ferroptosis in E1A;HRas^G12V;H11^Cas9 MEFs. (A) PI cell viability and lipid peroxidation analysis on E1A;Hras^G12V MEFs treated with erastin2 (era2) ± ferrostatin-1 (fer-1) in 5% O₂. n = 2 cell lines/sgRNA. Viability data are mean ± SD; *, P < 0.05, one-way ANOVA, Bonferroni’s multiple comparison test. Peroxidation data are mean percent FL1⁺FL2⁺ cells ± SD in 2–3 cell lines/sgRNA. *, P < 0.05, Student’s t test. (B) Experimental workflow for ferroptosis assays in 21% O₂. (C) Representative IncuCyte images of E1A;Hras^G12V MEFs in 21% O₂ treated for 8 h with era2 ± fer-1. Live cells are in red, and dead cells are in green. Scale bar, 50 μm. (D and E) Cell death in response to era2 (D) or era2 and 1 µM fer-1 (E) in 21% O₂. (F) Dose–response curves of era2-treated sgRNA-targeted E1A;Hras^G12V;H11^Cas9 MEFs at different time points in 21% O₂. (G) EC₅₀ and confidence interval (CI) values for era2-treated E1A;Hras^G12V MEFs in 21% O₂. (H) Basal lipid peroxidation in 5% and 21% O₂. Data are the mean percentage of FL1⁺FL2⁺ cells ± SD of 3 cell lines/sgRNA; **, P < 0.05, Student’s t test. For D–G, data are mean ± SD of 3 cell lines/sgRNA in three independent replicates. For EC₅₀ doses, the dose parameter was log transformed, and the data were fitted with a sigmoidal four-point curve with Hill slope constrained to 1 using Prism 7 (GraphPad).