Loss of p53 enhances stress fiber formation in E1A;Hras^G12V;H11^Cas9 MEFs. (A) Enrichr analysis of 569 p53-bound and regulated genes identified in RNA-seq analysis of E1A;Hras^G12V MEFs in 5% O₂. Overlap (hits/total genes in category) and adjusted P values are shown. (B) Heatmap showing expression of p53-dependent actin-related genes identified through Enrichr analysis of the E1A;Hras^G12V MEFs RNA-seq dataset. (C) Representative images of F-actin structures stained with phalloidin in E1A;Hras^G12V MEFs. DAPI marks nuclei. Scale bars, 25 µm and 5 μm (inset). (D) Quantitation of sgNTC- and sgp53-targeted E1A;Hras^G12V MEFs with 0–5 or >5 stress fibers/cell. Data represent the mean ± SD of n = 3 cell lines/sgRNA. ****, P < 0.0001, two-way ANOVA, Tukey’s multiple comparison test. (E) Total F-actin quantitation by phalloidin staining in sgRNA-targeted E1A;Hras^G12V MEFs. RFU, relative fluorescence units. Dots represent the total phalloidin fluorescence from one cell, with 35–110 cells analyzed per cell line. n = 3 cell lines/sgRNA. ns, not significant (P > 0.05), nested one-way ANOVA, Tukey’s multiple comparisons test. For A–E, all experiments were performed in 5% O₂.