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. 2022 Jun 22;13(6):563. doi: 10.1038/s41419-022-05003-4

Fig. 3. Palmitic acid drives cell fate via downregulation of p53.

Fig. 3

PA efficiently downregulates the expression of p53 and Mdm2. Western blot of total HepG2 cell lysate from control vehicle (BSA) and 0.5 mM PA in three different time points (12, 24, and 48 h). 0 h lysate put as a healthy cell condition and immunoblotted against p53 and Mdm2 (A). GAPDH was used as a loading control. Proteins were quantified as a normalized fold change density compared to individual BSA-treated conditions of a particular time point (B, C). Ordinary one-way ANOVA followed by Tukey’s multiple comparisons test was applied for statistical analysis. USP7 stabilizes p53 through its deubiquitinating activity. Western blot analysis of HepG2 cells transiently expressing WT-USP7 were treated with 0.5 mM PA for 24 h showing the dynamic expression pattern of Mdm2 and p53. The overexpression of USP7 rescued declined expression of p53 through Mdm2 independent manner (D). Pre-silencing of USP7 in 0.5 mM PA-treated HepG2 cells for 24 h were subjected to Mdm2 and p53 expression analysis through western blot. The knockdown efficiency completely destabilizes the Mdm2 and partially stabilizes p53 that is rescued from Mdm2, E3 ligase-dependent degradation (E). Further, western blot analyses of Mdm2 and p53 were performed in PA-treated HepG2 cells prior exposed to pharmacological inhibitors of USP7 for 24 h. Pre-treatment of both (P22077 and P5091; 10 and 20 µM) inhibitors showed rapid stabilization of p53 followed by a reduction in Mdm2 expression in a concentration-dependent manner upon 0.5 mM PA exposure for 24 h in the HepG2 cells (F). Consistently, overexpression of catalytic inactivated mutant (C223S-WT-USP7) fails to recover the p53 stabilization irrespective of Mdm2 de-stability in 24 h PA treated HepG2 cells (G). PA regulates G2/M checkpoints recovery. HepG2 cells were harvested upon PA (0.5 mM) exposure. The cell cycle analysis was performed on incubation of cells with propidium iodide (50 µg/ml) for 30 min in dark (H). The percentage of cell distribution through all phases was presented in the bar graph (I). Cell cycle distribution mapping was performed every 12 h interval till up to 48 h after post-treatment of PA. Two-way ANOVA followed by Tukey’s multiple comparisons test was applied for statistical analysis. Error bar represents mean ± SEM; n ≥ 3, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, and ns = non-significant.