Fig 2: Effects of antioxidants on SeC-induced cell death and the Nrf2 pathway.A Intracellular ROS levels (n = 7) were measures by fluorescent reader after SeC treatment with or without antioxidants (NAC and GSH). B cell viability (n = 8) of WiDr cells exposed to SeC with or without antioxidants (NAC and GSH) pretreatment. The values were presented as average ± SD from seven or eirht independent repeats. C Levels of phosphorylated Nrf2 and Nrf2 regulated proteins in WiDr cells after SeC treatment and GSH. GAPDH was used as loading control (n = 3) (*P < 0.05, **P < 0.01, ***P < 0.001, compared with solvent control, ##P < 0.01, ###P < 0.001, compared with SeC-treated cells without antioxidant pretreatment, significance was determined by one-way ANOVA). The values were presented as average ± SD from three independent repeats.

Fig 3: Model of heme-triggered DNA damage and cytotoxicity in HCEC versus CRC cells and role of HO-1.a Heme iron is taken up into HCEC, where it catalyzes the formation of reactive oxygen species (ROS) and induces oxidative DNA lesions as well as DNA strand breaks (indicated by red asterisks). This finally results in cytotoxicity, which is more prominent in HCEC than in CRC cell lines. In contrast, internalized inorganic iron causes little ROS production and DNA damage, and only slightly impairs cell viability. Heme-dependent ROS formation activates the transcription factor Nrf2, which shuttles from the cytoplasm to the nucleus, where it drives the transcription of its target genes such as heme oxygenase-1 (HO-1). This enzyme degrades heme to Fe2+, carbon monoxide (CO) and biliverdin (BV). Concomitant to HO-1 induction, ferritin heavy chain gene (FtH) is upregulated by hemin. Genetic abrogation of HO-1 by siRNA or its pharmacological inhibition by ZnPP potentiated heme-induced ROS, DNA damage and cell death, strongly suggesting that heme iron, and not its breakdown product Fe2+, initiates ROS formation and thus DNA damage induction. b Internalization of heme iron and inorganic iron in CRC cells. Similar to HCEC, inorganic iron causes little ROS production and DNA damage, and only slightly impairs viability in CRC cells. Hemin is taken up differentially into CRC cells (Caco-2 > HCT116), causing less ROS formation and oxidative DNA damage as in HCEC. Furthermore, CRC cells are in general more resistant against hemin-triggered cytotoxicity and HO-1 abrogation moderately affects cell survival in the presence of high heme concentrations. This figure was created using Servier Medical Art templates, which are licensed under a Creative Commons Attribution 3.0 Unported License; https://smart.servier.com.

Fig 4: TIGAR physically interacts with NRF2 and promotes NRF2 nuclear translocation. (A) MCF-7 cells were transfected with TIGAR or control siRNA, after 3 days, cells treated with H2O2 or vehicle for another 6 h, subcellular localization of TIGAR were examined by IF confocal microscopy. Cells were immunostained with NRF2 antibody (green). The nucleus is stained with DAPI (blue). Merged images (Merge) are shown. The scale bar represents 20 µm. (B) Subcellular localization of V5 tagged TIGAR and NRF2 were examined by IF confocal microscopy in A549 vector control cells or nuclear localization signal (NLS) linked TIGAR overexpressing cells. Cells were immunostained with V5 antibody (red) and NRF2 antibody (green). The nucleus was stained with DAPI (blue). Merged images (Merge) are shown. The scale bar represents 20 µm. (C) Whole cell lysates from MCF-7-TamR cells were prepared, and IP was performed with either anti-NRF2 antibody (top) or anti-TIGAR antibody (bottom) followed by IB with indicated antibodies. (D) Top left, schematics of His-tagged human NRF2 protein with numbers indicating boundary amino acid number at the indicated domains. GST pulldown assays were performed with GST-wild (WT) or mutant (TM) TIGAR and full length His-tagged NRF2 or its deletion forms. His-p97/VCP was used as a negative control.

Fig 5: 11aOH-KA upregulates Nrf2 pathways. B16F10 cells were treated with the indicated concentrations of 11aOH-KA or andrographolide (AG) for 24 h. The cells were recovered into sample tubes, and melanin contents were evaluated by photos (A) and optical density after lysis with NaOH (B). (C) B16F10 cells that had been transformed with the Nrf2 reporter (OKD48-luc) were treated with 11aOH-KA or AG for 24 h. Means and S.D. are shown (n = 3). *: p < 0.05. (D) The cells were lysed with SDS sample buffer without the reducing agent mercaptoethanol and used for Western blot analyses. (E) Awf (400 or 1200 µg mL-1) and Acf (300 or 900 µg mL-1) were used as stimulants. (F) B16F10 cells that had been precultured with or without 10 µM of 11aOH-KA for 24 h were treated with H2O2 for a further 24 h. Cell variability was examined by the Cell Counting kit (WST-8). (n = 4) ** p < 0.01. (G) The same experiments were performed with 11aOH-KA (10 µM), Awf (1260 µg mL-1), and Acf: (888 µg mL-1) in the presence of 200 µM H2O2. Gray and black bars indicate without H2O2 treatment and with only H2O2 treatment, respectively.