Gene expression analysis of sgNTC and sgp53(1) E1A;Hras^G12V;H11^Cas9 MEFs in physiological oxygen. (A) Loss of p53 induces pleiotropic effects in E1A;Hras^G12V MEFs in 5% O₂. (B) Schematic of RNA-sequencing pipeline of E1A;Hras^G12V MEFs in 5% O₂. n = 3 sgNTC and sgp53(1) targeted cell lines. (C) Volcano plot of P value versus fold-change expression of the 2,520 p53-regulated genes identified in RNA-seq analysis. Dots represent genes, and red dots indicate genes with a q value ≤0.05 and a fold-change ≥1.5. (D) Heatmap showing expression of well-characterized p53 target genes identified in RNA-seq analysis of sgNTC and sgp53(1)-targeted E1A;Hras^G12V MEFs (q value ≤0.05; no fold change cutoff). (E) Scheme for identifying direct p53 target genes by overlapping E1A;Hras^G12V RNA-seq data and p53 ChIP-sequencing data from primary MEFs treated with acute DNA damage (Kenzelmann Broz et al., 2013). (F–I) Heat maps showing gene expression signatures in sgNTC and sgp53(1)-targeted E1A;Hras^G12V MEFs for apoptosis (F), genomic fidelity/DNA repair (G), metabolic regulation (H), and migration and invasion (I) identified through literature analysis of the 226 genes that were p53-bound and >1.5-fold induced or repressed in E1A;Hras^G12V MEFs. Bolded genes were not previously identified as direct p53 targets in mouse or human in a meta-analyses of p53 expression profiling datasets (Fischer, 2019).