Fig. 2: Liver p53 engages Cyp2a5/CYP2A6 to support redox control during CCl4-mediated liver regeneration.

Staining (A) and quantification (B) for malondialdehyde (MDA) and oil-red-O (ORO) in Albumin-Cre; p53^WT/WT (WT) and Albumin-Cre; p53^FL/FL (FL) mice at 8 h after CCl₄ treatment. Scale bars 10μm. Representative of N = 3 WT and N = 4 FL mice. Data presented as mean ± SEM and analysed using two-tailed t-tests and the Sidak–Bonferroni method to account for multiplicity of tests. ns not significant. C Clustering analysis of significant differentially expressed genes (adjusted p < 0.05) from RNA-seq analysis between Albumin-Cre; p53^WT/WT (WT) and Albumin-Cre; p53^FL/FL (FL) mice at 8 h after CCl₄ treatment. Samples from N = 3 WT and N = 4 FL mice included in analysis. Positive Z-score values correspond to genes enriched in livers of mice of the genotype indicated in the sample name. Cyp2a5-associated cluster as indicated. For further information, see materials and methods. IHC staining (D) and quantification (E) of CYP2A5 in Albumin-Cre; p53^WT/WT (WT) and Albumin-Cre; p53^FL/FL (FL) mice at indicated times (hours) after CCl₄ treatment. Scale bars 20 μm. Quantification from N = 4 untreated (0 h) mice/group, N = 5 mice/group at 24 h, N = 7 mice/group at 48 and 72 h, and N = 3 mice/group at 168 h. Data presented as mean ± SEM and analysed using two-way ANOVA with Holm–Sidak’s multiple comparisons test and multiplicity-adjusted p values. ***p < 0.001, ****p < 0.0001. RT-qPCR analysis of expression of TP53, CDKN1A and CYP2A6 relative to ACTIN in HepG2 (F) and SK-Hep-1 (G) cells treated with siRNA against TP53, CYP2A6 (2A6), or non-targeting control (NT) 96 h prior to analysis and additionally treated with either DMSO control (UT), CCl₄ (4 mM), or with Nutlin (10 μM) for 24 h prior to analysis. N = 3 independent samples/condition. Data presented as mean ± SEM and analysed using two-way ANOVA with Holm-Sidak’s multiple comparisons test and multiplicity-adjusted p values. *p < 0.05, **p < 0.01, ****p < 0.0001. Measurement of cellular ROS levels relative to baseline using the CellROX fluorescent probe in HepG2 (H) and SK-Hep-1 (I) cells treated with non-targeting control (NT) or CYP2A6 (2A6) siRNA for 96 hours and additionally treated with either DMSO control (UT) or with CCl₄ (4 mM) in DMSO for 24 h prior to analysis. N = 5 independent HepG2 and N = 3 SK-Hep-1 samples/condition. Data presented as median fluorescent intensity (MFI) ± SEM relative to untreated NT cells and analysed using two-way ANOVA with Holm–Sidak’s multiple comparisons test and multiplicity-adjusted p values. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Measurement of cellular ROS levels relative to baseline using the CellROX fluorescent probe in HepG2 (J) or SK-Hep-1 cells (K) treated with non-targeting control (NT) or CYP2A6 (2A6) siRNA for 96 h and additionally treated with either DMSO control (UT) or with cumene hydroperoxide (10 μM) (CH) for 24 h prior to analysis. N = 3 independent samples/condition in HepG2 cells and N = 4/condition in SK-Hep-1 cells. Data presented as median fluorescent intensity (MFI) ± SEM relative to untreated NT cells and analysed using two-way ANOVA with Holm–Sidak’s multiple comparisons test and multiplicity-adjusted p values. **p < 0.01, ***p < 0.001, ****p < 0.0001.