Fig 2: Autophagy regulated OS and senescence in PTCs of Aldo-induced rats. (A) Senescence-associated-galactosidase (SA-ß-Gal) Staining in rat renal cortex (n = 6). SA-ß-Gal is labeled by bright-blue in the PTCs. (B) Bar graph indicating the percentage of SA-ß-Gal positive cells per field in tubular. (C) DHE staining of rat kidney sections (n = 6). (D) Bar graph indicating the mean DHE intensity per field in rat tubular cells. (E) Immunohistochemical staining for NOX4 in rat kidney tissues from various groups, as indicated (n = 6). (F) Bar graph indicating NOX4 immunoreactivity per field in rat tubular cells. (G) Western blot analysis revealed the expression of NOX4, p21, and GAPDH proteins after various treatments in rats (n = 3). (H) Graphical presentation shows the relative abundance levels of NOX4 and p21 after normalization with GAPDH (n = 3). #p < 0.05 vs. normal control, *p < 0.05 vs. Aldo alone.

Fig 3: The IC50 curve shows the cytotoxicity assay of (a) HGC-27 and (d) MGC-803 cell lines treated with Erastin. The flow cytometry and fluorescence microscope plots verify the different ROS levels of (b, c) HGC-27 and (e, f) MGC-803 cell lines treated with Erastin. The boxplots indicate the different mRNA expression levels of these 10 FDEGs in (g) HGC-27 and (h) MGC-803 after being treated with 10 µM Erastin by real-time PCR. (i) The plot detects the different protein expression levels of hub FDEGs (SP1, KEAP1, NOX4, AIFM2, and GPX4) in HGC-27 and MGC-803 after being treated with 10 µM Erastin by western blot. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Fig 4: Functional role of Nox4 in the regulation of vascular dysfunction by docetaxel in vivo.(A) Schematic design of in vivo studies of the effects of docetaxel on vascular endpoints. Nox4–/– and WT mice were injected i.p. with docetaxel (10 mg/kg) or placebo (solvent) every 5 days for 3 weeks. (B) Effect of docetaxel on blood pressure (BP) in C57BL/6J mice by telemetry. Data are expressed as the mean ± SEM. *P < 0.05 versus WT; 2-way, repeated-measures ANOVA. (C) Systolic blood pressure by tail-cuff plethysmography in Nox4–/– and WT mice treated with docetaxel or placebo (n = 10–14/group). Data are expressed as the mean ± SEM. #P < 0.01 versus baseline; **P < 0.01 versus WT or docetaxel-treated Nox4–/– mice; 2-way, repeated-measures ANOVA with Tukey’s test. (D) Endothelium-dependent vasorelaxation to ACh (1 nM–10 µM) and endothelium-independent vasorelaxation to SNP (1 nM–10 µM) in mouse aortas (n = 9–14/group). Data are expressed as the mean ± SEM. ***P < 0.001 versus WT; *P < 0.05 versus Nox4–/– mice treated with docetaxel; 2-way, repeated-measures ANOVA with Tukey’s test. (E) H2O2 production using Amplex Red in aortas from Nox4–/– and WT mice treated with docetaxel or placebo (n = 9/group). Data are expressed as the mean ± SEM. **P < 0.01 versus Nox4–/– mice treated with docetaxel; *P < 0.05 versus WT; 2-way ANOVA with Bonferroni’s test. (F) LGCL (5 µM) in aortas (n = 8–9/group). Data are expressed as the mean ± SEM. **P < 0.01 versus WT; 2-way ANOVA with Bonferroni’s test. (G) Representative images showing superoxide production in mouse aortas using DHE fluorescence. Scale bars: 100 µm. (H) Nox4 mRNA expression in mouse aortas (n = 5/group). Data are expressed as the mean ± SEM. ****P < 0.0001 versus Nox4–/– mice treated with docetaxel; *P < 0.05 versus WT; 2-way ANOVA with Bonferroni’s test. (I) p-eNOS (Thr495) in Nox4–/– and WT mouse aortas treated with docetaxel or placebo (n = 5–6/group). Densitometric analysis was normalized to total eNos. Immunoblots represent 1 of 2 independent experiments and are summarized as the mean ± SEM. **P < 0.01 versus Nox4–/– mice treated with docetaxel; ***P < 0.001 versus WT; 2-way ANOVA and Bonferroni’s test.

Fig 5: Effect of luminal flow on Nox4 localization and activation in thick ascending limbs. Panels A and B: Effect of luminal flow on Nox4 localization in thick ascending limbs using an antibody against the carboxyl terminus of Nox4 (C-Nox4); (A) Representative immunofluorescent confocal images showing C-Nox4 staining (left panel) and same nuclei counterstained with POPO3 (right panel). (B) Quantification using the ratio between nuclear and cytoplasmic intensities (N/C intensity; n = 5 and 6, for no flow vs. flow, respectively). Panels C and D: Effect of luminal flow on Nox4 localization in thick ascending limbs using an antibody against the amino terminus of Nox4 (N-Nox4); (C) Representative immunofluorescent confocal images showing N-Nox4 staining (left panel) and same nuclei counterstained with POPO3 (right panel). (D) Quantification using the ratio between nuclear and cytoplasmic intensities (N/C intensity; n = 6 for each group). Nuclei are outlined for better identification.