Fig. 4: Hepatocyte-specific neddylation deficiency induces oxidative stress and suppresses metabolic function.
a Venn diagrams showing comparisons of numbers of differentially expressed genes (DEGs, 1.0 ≤ LogFc ≤ −1.0; Pad < 0.05) in livers from male P10 Nae1f/f; Alb-Cre + (LKO) and D21 AAV-Cre (Cre) mice as compared to Nae1f/f (WT) and AAV-GFP (GFP) counterparts, respectively. The intersection of the two circles shows DEGs in both LKO and Cre livers. b KEGG pathway enrichment analysis of DEGs significantly up-and downregulated in both LKO and Cre livers (n = 4 per group). c Scatter plots comparing DEGs of D21 Cre vs GFP with DEGs of GSE38941 (t = 27.89, df = 3304, 95% confidence intervals of 0.41–0.46) or GSE96851 (t = 29.56, df = 3304, 95% confidence intervals of 0.43–0.48), respectively. Cor.test() function in R was used. d Total ROS levels were measured by incubating liver homogenates with a DCFH-DA probe and expressed as AU (n = 6 per group, *P = 0.0053). e GSH (*P = 0.0075) and GSSG (**P = 0.0031) levels normalized to liver weights and GSSG/GSH ratio (**P = 0.0035) (GFP: n = 7; Cre: n = 8). Male D21 GFP and Cre mice and two-tailed unpaired t-tests were used in (d, e). f–l Primary hepatocytes (PH) were isolated from the livers of D12 male GFP and Cre mice. f Western blot (n = 2 per group); g MitoSOX staining and quantification of MitoSOX-positive cells from 10 images. Scale bar: 100 μm. **P < 0.0001. h Total GSH levels normalized to protein levels (n = 6 per group). *P = 0.0072. i Oxygen consumption rate (OCR) was detected using a Seahorse XF Cell Mito Stress Test. Basal, maximal (Max.), coupled, and uncoupled mitochondrial (Mito.) respirations were shown in j. **P < 0.005. k Intracellular ATP contents normalized to protein levels (n = 6 per group). *P = 0.0171. l Cell viability was determined by MTT assays and expressed as AU (n = 6 per group). **P = 0.0018. Representative data from three biologically independent experiments were shown. Two-tailed Unpaired t-tests in (g, h, k, l). Multiple unpaired t-tests with the Holm-Sidak method in (j). GFP (black), Cre (red). All quantitative data were presented as mean ± SEM. Source data are provided as a Source Data file.
