Fig 2: SAAG, SA and AG protected against H2O2‐induced neurotoxicity in PC12 cells; A, Viability of PC12 cells treated with SAAG, SA and AG (n = 5); B, Viability of PC12 pre‐treated with drugs and then followed with H2O2 treatment (n = 6); C, ROS level (n = 6); D, Nitric Oxide level (n = 4); E, GSH level (n = 4); F, TrxR activity (n = 3); G, Nuclear Nrf2 activation (n = 4); H, Cell viability by SAAG was abolished by Aur and ML385; (n = 6), I, Cellular ROS level enhanced by Aur and ML385 (n = 6); J, NO content increased by Aur and ML385 (n = 4); To reveal the role of SA and AG in SAAG, 0.6 mg/mL AG and 10 mg/mL SA was compared with 20 μL/mL SAAG in H2O2‐induced cell injury model, since every millilitre of SAAG contains 30 mg AG and 0.5 g SA according to its instructions; K, Schematic diagram illustrating that SAAG simultaneous enhanced the Trx, GSH and Nrf2 systems against cerebral I/R injury through ASK1‐JNK/p38 cascades. # P < .05 compared with the control group, *P < .05 compared with the model group, and ∆ P < .05 compared with the SAAG group

Fig 3: Shh and Nrf2 expression predicts OS in HNSCC. (a) Shh and Nrf2 expression in matched normal and tumor samples (Mann–Whitney U test, p < 0.0001) (b) Shh and Nrf2 expression stratified by tumor stage (one-way ANOVA, p < 0.0001, p < 0.05, p < 0.01). (c) Kaplan–Meier survival curves comparing the OS for patients with HNSCC grouped as Shhhigh and Shhlow (log-rank p < 0.001), and Nrf2high and Nrf2low expression (log-rank p < 0.0001; high: Score3/Score2; low: Score1/Score0 expression groups). Comparison was made between high and low expression groups. Below each graph showed the Cox proportional hazard ratio in a multivariate analysis (p < 0.01 and p < 0.001).ANOVA, analysis of variance; HNSCC, head and neck squamous cell carcinoma; OS, overall survival.

Fig 4: Mini-GAGR induces phosphorylation and nuclear localization of Nrf2 in a PKC- and FGFR1-dependent manner. A, mouse cortical neurons (E17, DIV14) were treated with 1 μm mini-GAGR (mini) for 3 h and processed for immunoblotting (p-Ser-40-Nrf2 and Nrf2). The band densities of phospho-Nrf2 (100 kDa) (B) and total Nrf2 (80 kDa) (C) were quantified by ImageJ to obtain average density ± S.E. (error bars) of p-Nrf2 (p < 0.001, n = 3 different embryo batches), Nrf2 protein concentration (n = 3 different embryo batches) (Student's t test, two-tailed). D, primary cortical neurons (E17, DIV14) were pretreated with either vehicle or 3 nm Stau (PKC inhibitor) for 2 h and treated with either vehicle (control) or 1 μm mini-GAGR for 3 h prior to the measurement of PKC activity (n = 8 different embryo batches): control versus mini-GAGR (mini) (p = 0.008), control versus Stau (p = 0.017), control versus Stau + mini-GAGR (p = 0.047), mini-GAGR versus Stau + mini-GAGR (p < 0.001) (one-way ANOVA (F(3, 60) = 14.631, p < 0.001, ANOVA, and Bonferroni's multiple-comparison test). E–G, primary cortical neurons (E17, DIV14) were pretreated with vehicle, 3 nm staurosporine, or 50 nm PD173074 (FGFR1 inhibitor) for 2 h and treated with 1 μm mini-GAGR for 3 h prior to immunoblotting (p-Nrf2 and Nrf2). E, the band densities of antioxidant enzyme proteins were quantified by ImageJ to obtain average density ± S.E. for bar graphs: control versus mini-GAGR (n = 4 different embryo batches, p < 0.01 (F (5, 12) = 9.4859, p < 0.001, ANOVA)). F and G, primary cortical neurons (E17, DIV14) were pretreated with vehicle, 3 nm staurosporine, or 50 nm PD173074 for 2 h and treated with 1 μm mini-GAGR for 3 h prior to immunocytochemistry (Nrf2 (green; Alexa Fluor 488), βIII-tubulin (red; Alexa Fluor 594), DAPI). F, the fluorescence intensities of nuclear Nrf2 in images were quantified using Metamorph to calculate the average intensity ± S.E. of nuclear Nrf2 staining for bar graphs (n = 100–123 cells): control versus mini-GAGR, Stau, Stau + mini-GAGR, PD, PD + mini-GAGR (p < 0.001); mini-GAGR versus Stau + mini-GAGR, PD + mini-GAGR (p < 0.001) (F(5, 877) = 137.67, p < 0.001, ANOVA). *, p < 0.05; **, p < 0.01; ***, p < 0.001, one-way ANOVA and Bonferroni's multiple-comparison test). Data are expressed as mean ± S.E. (scale bar, 30 μm). (Of note, because the total number of measurements per condition was over 100, scatter plots were not used for graphs.)

Fig 5: Inhibition of Nrf2 radiosensitizes breast cancer cells by inducing apoptosis and suppressing BCSC population after radiation treatment. (A) BCSC population measured by ALDH activity using flow cytometry in shNrf2 MCF-7 cells and mammospheres. (B) Phase-contrast images depict the effect of fractionated and acute doses of radiation on sphere formation in shNrf2 MCF-7 cells. The bar graph represents mammosphere formation efficiency for the same. (C) Expression of stem cell markers, i.e., SOX2, KLF4 and NANOG, was analyzed in shNrf2 MCF-7 cells and mammospheres by Western blotting, GAPDH is used as loading control. All values are given as the mean ± SE, *** p < 0.001 vs. fractionated-dose-irradiated shNrf2 cells. (D) The image demonstrates isolated tumors of the xenograft derived from shNrf2 MCF-7 control and irradiated cells. (E) ALDH activity was measured in shNrf2-derived tumors. (F) The representative images show a decrease in the colony formation of shNrf2 MCF-7 cells and mammospheres upon fractionated dose radiation treatment. (G) The bar graphs depict the percentage of apoptotic cells in shNrf2 MCF-7 cells and mammospheres. All values are given as the mean ± SE, * p < 0.05, ** p < 0.01; vs. fractionated dose irradiation shNrf2 cells. All images are representative of three independent experiments.