Fig. 2: Loss of caspase-2 leads to a similar increase in ferroptosis in mut-p53 cancer cells.

a Immunoblot analysis of caspase-2 expression in H1299p53^R273H cells following CRISPR/Cas9 editing of CASP2 (CASP2^−/−) vs. control cells (Cas9). Vinculin is shown as the loading control. b Mean IC50 values (μM) in H1299p53^R273H Cas9 and H1299p53^R273H -CASP2^−/− cells after 72 h treatment with erastin. c Representative bright field images from live-cell imaging at the indicated time points of Cas9 and CASP2^−/− H1299p53^R273H cells treated with erastin (2 μM). Right-hand panels display dead cells stained red with propidium iodide (PI + ). Scale bar = 50 μm. d Representative images of crystal violet stained colonies of Cas9 or CASP2^−/− H1299p53^R273H cells treated with erastin or vehicle for 12 h, re-seeded and cultured over 10 days (left). Quantitation of crystal violet stained cell colonies using Cell Profiler software and represented as the area covered by the colonies (right). e Lipid peroxidation analysis by flow cytometry using C11-BODIPY post RSL3 (40 nM) or vehicle treatment for 18 h in H1299p53^R273H Cas9 and H1299p53^R273H -CASP2^−/− cells. f Total intracellular glutathione (GSH + GSS pmol/10⁶ cells), as determined by GR re-cycling assay after 12 h erastin (2 μM) treatment in H1299p53^R273H Cas9 and H1299p53^R273H -CASP2^−/− cells. (b, d right, e, f) Data represented as mean ±s.e.m. from three independent experiments. Unpaired t-test and one-way ANOVA with Tukey’s post hoc test were used to estimate significant differences in b, f, and d, e respectively. P-values are indicated with *P < 0.05, **P < 0.01, and ***P < 0.001.