Fig 2: Nrf2 activation effect of K22 on H9c2 cells. (A) Cells were treated with K22 at various concentrations for 12 h. (B) Cells were exposed to K22 (10 μM) at various time points. qRT-PCR assay was applied to analyse the mRNA levels of Nrf2 and Nrf2-targeted gene. GAPDH was used to normalise the expression of these genes with DMSO-treatment group used as the blank control. (C) Cells were treated with K22 at various concentrations for 12 h. (D) Cells were exposed to K22 (10 μM) at various time points. Western blot assay was applied to determine the protein levels of Nrf2 and Nrf2-regulated proteins. (E&F) Statistics of the protein expression levels of Nrf2, HO-1 and NQO1. GAPDH was used as the internal control. Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 vs. Control group.

Fig 3: The expression levels of Nrf2 and Nrf2-regulated genes after treatment with Nrf2 siRNA and K22. H9c2 cells were incubated with Nrf2 siRNA (50 nM), K22 (10 μM), or Nrf2 siRNA (50 nM) plus K22 (10 μM). qRT-PCR was then used to quantify the expression levels of Nrf2, HO-1 and NQO1 genes. ***p < 0.001.

Fig 4: hPMSC-Exo upregulates the expression of Nrf2 via PTEN/PI3K axis in CD4+ T cells from D-gal-treated mice. (A) The protein expressions of PTEN/PI3K-Nrf2 axis and its downstream target genes were evaluated by Western blot. (B) The protein level of PTEN. (C) The ratio of p-PI3K/PI3K. (D) The ratio of p-Akt/Akt. (E) The protein level of nuclear Nrf2. (F) The protein level of NQO1. (G) The protein level of HO-1. Data represent the mean scores ± SEM of at least three independent experiments. **p < 0.01 vs. control group; ## p < 0.01 vs. PBS group.

Fig 5: Nrf2 is an endogenous suppressor of IFN-β in inflammatory macrophages(A) IFN-β, IL-6, and TNF protein levels in LPS-stimulated Nrf2-KO and Keap1-KD compared to WT cells (n = 3 biological replicates).(B) Ifnb and Ifit2 mRNA levels in LPS-stimulated Nrf2-KO and Keap1-KD compared to WT cells (n = 5-6 biological replicates).(A-B) Data are mean ± SEM p value determined by unpaired t test, corrected for multiple comparisons by Holm-Sidak test.(C) IFN-β in Poly(I:C) stimulated Nrf2-KO and Keap1-KD compared to WT cells (n = 3 biological replicates). Data are mean ± SEM p value determined by two-tailed unpaired t test.(D) Ifnb and Ifit2 mRNA levels in WT, Nrf2-KO, and Keap1-KD cells that had been pre-treated for 1 h with the Nrf2 activator TBE-31 (30 nM) and stimulated with LPS (100 ng/mL) for a further 4 h (n = 3 biological replicates).(E) Ifnb and Ifit2 mRNA levels in WT, Nrf2-KO, and Keap1-KD cells that had been pre-treated for 24 h with the Nrf2 activator TBE-31 (20 nM) and stimulated with LPS (100 ng/mL) for a further 4 h (n = 2-3 biological replicates).(D-E) Data are mean ± SEM p value determined by one-way ANOVA, corrected for multiple comparisons by Tukey statistical test.(F) Nqo1 mRNA levels in WT, Nrf2-KO, and Keap1-KD cells that had been pre-treated for 24 h with the Nrf2 activator TBE-31 (20 nM) (n = 2-3 biological replicates). Data are mean ± SEM p value determined by a two-tailed unpaired t test. p <0.05∗; p <0.01∗∗; p <0.001∗∗∗.