Fig. 8: FoxO3 deficiency-induced hepatocyte proliferation exhibits a strong correlation with Nox4 downregulation.

a RNA-seq identified 4 overlapping downregulated genes in AKO mice pre- and post-PH compared with control mice. G1, control mice pre-PH; G2, control mice post-PH; G3, mutant mice pre-PH; G4, mutant mice post-PH. b qPCR validation of the downregulated expression of Nox4 in FoxO3-knockout mice compared with control mice before and after PH (n = 8 mice per group). c Downregulation of Nox4 expression in FoxO3-deficient NCTC1469 cells (n = 5 per group). d Upregulation of Nox4 expression in FoxO3-overexpressing NCTC1469 cells (n = 5 per group). e Schematic of AAV-Nox4 injection in adult AKO mice for 2 weeks, followed by primary hepatocytes isolation at 0 dpH or PH performance and EdU injection at 2 dpH. f, g Isolated primary hepatocytes at 0 dpH were treated with HGF (40 ng/ml) for 4 days in MHGM, followed by Western blotting assay (f, n = 3 mice) and EdU/HNF4α double staining (g, n = 6 mice). h qPCR validation of Nox4 expression in livers from AAV-NC and AAV-Nox4 mice (n = 3 mice). i Representative images (left) and quantification (right) of Ki67⁺ HNF4α⁺ cells in AAV-NC and AAV-Nox4 mice at 2 dpH (n = 6 mice). j Representative images (left) and quantification (right) of Ki67⁺ cells in liver at 2 dpH (n = 6 mice). k Representative images (left) and quantification (right) of EdU⁺ HNF4α⁺ cells in AAV-NC and AAV-Nox4 mice at 2 dpH (n = 6 mice). l Representative images (left) and quantification (right) of pH3⁺ HNF4α⁺ cells in livers at 2 dpH (n = 6 mice). m Representative images (left) and quantification (right) of mitotic hepatocytes in livers at 2 dpH (n = 6 mice). n Cell counting assay for primary hepatocytes isolated from livers at 2 dpH (n = 6). o Representative images of livers at 2 dpH. p The actual ratio of liver/body weight at 2 dpH (n = 6 mice). q and r Liver ALT (q) and AST (r) levels in AKO mice injected with AAV-NC and AAV-Nox4 at 2 dpH (n = 3 mice). All data are presented as the mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001 versus controls (two-way ANOVA test for b; Student’s t-test for c, d, f-r).