Fig 1: Quantification of NRF2 transcript and protein expression from whole tissue extracts. (A) Relative transcript expression of NRF2 and its target genes HMOX1, TXNRD1, and GPX2. Box plots show the median, maximum, and minimum of 2−ΔΔCt values derived from real-time qPCR. Each dot represents the mean value of an individual sample. (B) Western blot showing NRF2 protein expression in nuclear and cytoplasmic extracts of three representative AAA and three control samples. (C) Quantification of the Western blot analysis (n = 7 control and n = 11 AAA tissue extracts). Box plots show the median, maximum, and minimum of the relative protein expression after normalization as described in the methods section. Each colour circle represents the value of an individual sample (A,C) Data were analysed by Mann–Whitney test. *: p < 0.05, **: p < 0.01, ****: p < 0.0001, ns: not significant. (D) Correlation matrix visualizing the relationships between transcript expression of different genes in whole tissue extracts.
Fig 2: Comparison of baseline NFR2 protein levels (A) NFR2 activity (B) and NRF2 transcript expression without additional oxidative stress induction. (A) NRF2 protein levels were determined by ELISA and normalized to the total protein level of each sample. Data are shown as pg NRF2 per µg protein. Each color circle represents a normalized value derived from n = 4 AAA-SMC, n = 3 healthy VSMC samples and one immortalized cell line (IM), analysed in duplicate. (B) NRF2 binding activity to DNA was assayed from nuclear extracts by using a colorimetric NRF2 activity assay. Data were normalized to the protein content of the corresponding nuclear extracts. Each color circle represents a normalized value derived from n = 2 AAA-SMC, n = 3 healthy VSMC and one immortalized cell line (IM), analysed in duplicate. (C) NFE2L2 transcript expression was analysed by qPCR using specific primers. Data show the 2−(ΔCt) values. Each colour circle represents a normalized value of n = 2 AAA-SMC (AAA), n = 3 healthy VSMC (healthy), and one immortalized cell line (IM), analysed in triplicate. Bars represent the mean ± SEM. Data were statistically analysed by ordinary one-way ANOVA and Tukey’s multiple comparison test; ns: not significant. ** p < 0.01, *** p < 0.001, **** p < 0.0001.
Fig 3: Immunohistochemical analysis of αSMA, KEAP1, NRF2, and 8-OHdG in the media and intima of AAA tissue samples and healthy aorta (control). (A–D) Representative image details of n = 22 AAA and n = 9 healthy control tissue samples are shown. The white dashed lines and squares indicate the enlarged sections. (E) The entire available surface area of each specimen was used for the analysis. Box plots show the median, maximum, and minimum of percent positive staining normalized to the total media/intima area. Each colour circle represents a normalized value of an individual tissue sample. Data were statistically analysed by unpaired t test. *: p < 0.05, **: p < 0.01, ****: p < 0.0001. 1000 μm, 150 μm.
Fig 4: Response of AAA-SMC, immortalized iHAoSMC, and healthy VSMC against acute oxidative stress induced by exposure to H2O2. (A) Treatment scheme for induction of acute oxidative stress. Cell lines were treated with 10 nM of MitoQ or vehicle for 7 days before they were challenged with 100 µM H2O2 for 30 min. (B) NRF2 binding activity to DNA (antioxidative response element, ARE). Nuclear extracts were harvested immediately after H2O2 treatment and analysed by using an NRF2 activity assay. (C) Transcriptional expression of the NRF2 target gene HMOX1 (HO1), 6 h after H2O2 treatment. Data show the 2−ΔΔCt values derived from qPCR. (D) HO1 protein expression was analysed by ELISA. Protein extracts were harvested after 24 h of regeneration. (B–D) Data are derived from n = 2 individual AAA-SMC cultures, n = 3 individual healthy VSMC cell cultures, and one iHAoSMC line. Bars represent the mean ± SEM. Data were statistically analysed by two-way ANOVA and Tukey’s multiple comparison test; ns: not significant, *: p < 0.05, ** p < 0.01, ***: p < 0.001, ****: p < 0.0001.
from Cell Signaling Technology for NRF2 Control Cell Extracts