Fig 2: BMSCs exhibit increased apoptosis and oxidative stress along with reduced mitochondrial membrane potential in hypoxic condition. (a) Isolation and identification of BMSCs by detecting cell surface antigens via flow sorting. (b, c) Flow cytometry and (d, e) TUNEL staining for examining apoptosis of BMSCs under exposure to hypoxia for 6, 12, and 24 h. Bar, 20 µm. (f, g) JC-1 staining for detecting mitochondrial membrane potential of hypoxic BMSCs. Bar, 20 µm. (h–j) Immunoblotting of the activity of Keap1 and Nrf2 in hypoxia-exposed BMSCs.

Fig 3: CNPs counteract DDP-induced oxidative stress via activating Nrf2/Keap1 signaling pathway.a Western blot analysis of the Nrf2, Keap1, and DJ-1 levels in HK-2 cells after respective treatments with the vehicle, CNPs, DDP, and CNPs plus DDP. GAPDH served as a loading control. n = 2 independent experiments. b, c, d Relative mRNA expressions of Nrf2 (b), Keap1 (c), and DJ-1 (d) in the renal cortex from each group. n = 4 independent experiments. In b, P(DDP) = 0.0091, P(0.5 CNPs) = 0.015, P(1.5 CNPs) = 0.00026; in c, P(DDP) = 0.003, P(0.5 CNPs) = 0.0977, P(1.5 CNPs) = 0.0054; in d, P(DDP) = 0.0006, P(0.5 CNPs) = 0.0185, P(1.5 CNPs) = 0.00031. e Schematic diagram of the experimental setup to evaluate the role of Nrf2 by small interfering RNA (SiRNA) in vitro using western blot and qRT-PCR analysis. f Western blot analysis of Nrf2, clv-PARP and clv-caspase-3 levels in SiNrf2-transfected HK-2 cells. GAPDH served as a loading control. n = 2 independent experiments. g, h Relative mRNA expressions of HO-1 (g) and NOX2 (h) in the respective groups. n = 4 independent experiments. In g, P(siNC, DDP) = 0.0092, P(siNC, DDP+CNPs) = 0.0001, P(siNrf2, DDP) = 0.00046, P(siNrf2, DDP+CNPs) = 0.9958; in h, P(siNC, DDP) = 0.0241, P(siNC, DDP+CNPs) = 0.0382, P(siNrf2, DDP) = 0.0139, P(siNrf2, DDP+CNPs) = 1.000. i Schematic representation of the mechanism of CNPs in reducing ROS level. CNPs decompose DDP-induced H2O2, a predominant cellular ROS, into H2O and O2. Meanwhile, the minimally residual amounts of ROS activate the Nrf2/Keap1 signaling pathway. Specifically, Nrf2 moves into the nucleus and subsequently binds to the antioxidant response elements (ARE), leading to the upregulation of the antioxidant gene (HO-1) and downregulation of the pro-oxidant gene (NOX2). These gene regulations can further detoxify ROS. Each experiment was repeated three times independently. Data are presented as means ± SEM., *P < 0.05, **P < 0.01, ***P < 0.001, #P < 0.05, ##P < 0.01, ###P < 0.001, n.s., no significance; one-way ANOVA with multiple comparisons test. Source data are provided as a Source Data file.

Fig 4: Loss of Keap1 increases the Nrf2 transcriptional activity, increase cancer stem cell characteristics, and predictor of chemotherapeutic outcome in patients with HNSCC.A Cal33 cells were transfected with siRNA against Keap1, scrambled, and control for 96 h. Keap1 mRNA was assessed by quantitative RT-PCR. Results expressed as fold-change. B Cal33 cells were transfected as described in A and SOD1 mRNA was assessed by quantitative RT-PCR. C Cells were treated with Keap1 siRNA to knock down the Keap1 gene and assessed the cell viability 72 h after cisplatin treatment in the indicated concentrations in Cal33 cells. Data presented as mean SD of triplicate experiments. D Cell survival at 72 h after cisplatin treatment of indicated HNSCC patient’s primary tumor and HNSCC cell lines (*P < 0.05, **P < 0.01). E qRT-PCR analysis of Keap1 expression in control, Keap1 expressing SSC9 clone and parental SSC9 cells (***P < 0.001). F qRT-PCR analysis of Nrf2 target genes SOD1 and NQO1 in control, Keap1 expressing clone, and parental SSC9 cells (***P < 0.001). G Cell proliferation activity of Keap1 expressing clone, control, and parental SSC9 cells. H Cell survival at 72 h after cisplatin treatment in parental SCC9, mock-transfected and Keap1-expressing clones. I Relative number of tumorspheres generated by the indicated patient’s tumor cells and cell lines. J Relative number of tumorspheres in parental SCC9, mock-transfected, and Keap1-expressing clone (**P < 0.01). K Expression of CD44 in cisplatin-resistant (n = 13) and cisplatin-sensitive (n = 11) HNSCC patients. L Summary of the results for the CD44 expression analysis in the presence of Keap1 or Nrf2 mutations and/or Keap1 or Nrf2 protein expression in each case (n = 24). The number of aberrations in each case was represented as the aberration scores (0, 1, 2, and 3) and all 24 cases were assigned into two groups based on the aberration scores: a “high score group” (n = 13 as aberration score 2 and 3) and low score group (n = 11 as aberration score 1, and 0). M Kaplan–Meier disease-free survival curve for 24 patients was generated according to the aberration score. The high score group was significantly associated with shorter disease-free survival (Log-rank p < 0.0001).

Fig 5: Impaired autophagy can activate NFE2L2 through the SQSTM1-KEAP1 pathway in astrocytes. (A) Western blot analysis of nuclear NFE2L2 protein levels in astrocytes after treatment with autophagy inhibitor Atg7 siRNA and astrocyte activators TNF or H2O2. (B,C) Statistical results of nuclear NFE2L2 protein after treatment with TNF (B) or H2O2 (C) in Fig. 8A. Data are presented as the mean ± SD (N = 3). *p < 0.05, **p < 0.01; ns: no significance. (D,E) Western blot analysis (D) and statistical results (E) of the protein levels of LC3-II, SQSTM1, and NFE2L2 in astrocytes after treatment with H2O2 or 3-MA or rapamycin (Rapa). Data are presented as the mean ± SD (N = 3). *p < 0.05, **p < 0.01. (F) Effect of inhibition of SQSTM1 on the protein levels of KEAP1 and nucleus NFE2L2 in autophagy impaired astrocytes. (G) Statistical results of the protein levels of SQSTM1, KEAP1, and NFE2L2 in Fig. 8 F. Data are presented as the mean ± SD (N = 3). *p < 0.01, **p < 0.01; ns: no significance