Fig 1: Effect of increasing doses of AFB1 (i.e., 0.9 µM, 1.8 µM and 3.6 µM) on NQO1 mRNA expression and catalytic activity. (A) Gene expression data are reported as the mean ± SEM of logCPM relative to four biological replicates. (B) Catalytic activity is expressed as median optical density (OD) per minute per mg of total protein ± SEM of four biological replicates, each one tested in duplicate. Statistical analysis: one-way ANOVA followed by Bonferroni’s multiple comparisons test; *: p < 0.05, **: p < 0.01 and ***: p < 0.001, treated cells vs. control cells.
Fig 2: AFB1 induction of hepatotoxicity in BFH12 cells. Danger signals deriving from oxidative stress, as well as the DAMP, lead to TLR2 activation which triggers a downstream signaling cascade with the involvement of p38ß MAPK. In turn, p38ß MAPK allows the nuclear translocation of AP-1 and NF-kB transcription factors, thus leading the release of IL6 pro-inflammatory cytokine. In addition, p38ß MAPK induction negatively regulates the expression of NRF2-dependent cytoprotective genes (e.g., NQO1, UGT1A1 and ALDH1A1), while IL6 inhibited CYP2B6 mRNA expression.
Fig 3: LncRNA NEAT1 positively regulates NOQ1 expression in MDA-MB-231 cells. (A) SP600125 (SP), the JNK inhibitor, was used as 10 µM and the JNK activation was determined by western blot analysis of p-JNK at 6h (left panel). The expression of NQO1 or Nrf2 in radioresistant MDA-MB-231 cells after SP treatment was examined by western blot analysis (right panel). a-tubulin or GAPDH was used as the loading control. (B) The CSC activity of MDA-MB-231 (231-P) or radioresistant MDA-MB-231 (231-RR) cells was determined by tumorsphere cultivation. The numbers of formed tumorspheres were pictured (left panel) and counted (right panel) at Day 7 post-seeding. **, p< 0.01. (C) NEAT1 expression in 231-P or 231-R cells was determined by SYBR Green based quantitative RT-PCR. **, p< 0.01. (D to G) Inhibition of NEAT1 expression in 231-RR cells was performed by transfection of non-viral vector containing Cas9 and NEAT1 sgRNA sequence (sgNEAT1) and selected by puromycin. A Cas9-expressing plasmid was used as a negative control (Cas9). NEAT1 expression after puromycin selection was determined by qRT-PCR (D). NQO1 expression was detected by western blot (E). The inserted numbers indicated relative expression level after compared to Cas9 group. The NQO1 mRNA expression of Cas9- or sgNEAT1-transfected cells was determined by qRT-PCR (F). Cas9- or sgNEAT-transfected 231-RR cells were irradiated for 2 or 4 Gy and the fractional survivals of cells were determined by clonogenic assay and calculated by the linear-quadratic model (G). **, p< 0.01.
Fig 4: The effect of pretreatment time on Nrf2 signaling pathway activated by fucoxanthin in differentiated RPE cells. (a) Nucl-Nrf2 activity; (b) GCLC expression level; (c) GPx expression level; (d) TrxR expression level; (e) HO-1 expression level; (f) NQO1 expression level. (** p < 0.01 vs. control; ?? p < 0.01 vs. lutein).
Fig 5: The protein expression and activity of NQO1 is upregulated in radioresistant MDA-MB-231 cells. (A) Radiosensitivity of Hs578t, MDA-MB-231 (231-P) or the radioresistant MDA-MB-231 (231-RR) cells was determined by clonogenic assay. Data were presented as survival fractions which were calculated by the linear-quadratic model using GraphPrism software. **, p< 0.01; *, p< 0.05. (B) NQO1 protein expressions in Hs578t, 231-P, or 231-RR cells were determined by western blot. GAPDH was used as a loading control. (C) The NQO1 mRNA expression in 231-P or 231-RR cells was determined by qRT-PCR method and data were presented as relative expression level when compared to 231-P cells. (D) NQO1 activity in cellular protein preparation was determined as the described in Materials and Methods section. *, p< 0.05.
Supplier Page from Abcam for NQO1 Activity Assay Kit