Fig 2: Effects of MitoQ, MSU, TRX siRNA and TXNIP siRNA on TXNIP/NLRP3/IL-1ß axis and pro-fibrotic protein expression and apoptosis in HK-2 cells cultured under HG conditions. A: IF showing FN (Aa-c) and Collagen I (Ad-f) expression in HK-2 cells subjected to HG treatment following MitoQ treatment. B: WB analysis of Caspase-1, cleaved IL-1ß and IL-18 expression in HK-2 cells cultured under HG conditions and treated with different concentrations of MitoQ (50,100, 150 nM). C-H: ELISA of Caspase-1 (C), IL-1ß (D), IL-18 (E), TGF-ß (F) activity; TGF-ß expression (G); and relative apoptosis (H) in HK-2 cells subjected to HG conditions and treated with different concentrations of MitoQ (50, 100, 150 nM). I: Luminex analysis of IL-1ß, IL-18, FN and Collagen I expression in HK-2 cells subjected to an HG environment and treated with MitoQ, MSU or TXNIP siRNA. J, K: WB and densitometric analysis of NLRP3, IL-1ß, IL-18 and FN expression in HK-2 cells exposed to an HG environment and pretreated with MitoQ, TRX siRNA or TXNIP siRNA. Values are the mean ± SE; *P < 0.05 vs control group; #P < 0.05 vs HG group. n = 3.

Fig 3: Effects of MitoQ on renal NLRP3, TRX, TXNIP, Caspase-1 and IL-1ß expression in db/db mice.A: Renal expression of the proteins detected by iTRAQ-based quantitative proteomics analysis in db/m and db/db mice. Green represents down-regulation, red represents up-regulation, and black represents no change. The bar code (- 1–1) at the top shows the color scale of the log2 values. B: Immunohistochemical analysis of NLRP3 (Ba-c), TRX (Bd-f), TXNIP (Bg-i), Caspase-1 (Bj-l) and IL-1ß expression (Bm-o) in kidney tissues. (magnification × 200). C: Quantification of NLRP3, TRX, TXNIP, Caspase-1 and IL-1ß expression in tissues from the different groups. D-G: Analysis of the correlations between NLRP3 expression and TRX, TXNIP, Caspase-1 and IL-1ß expression: r (NLRP3/TRX) = -0.707 (D), r (NLRP3/TXNIP) = 0.695 (E), r (NLRP3/Caspase-1) = 0.674 (F) and r (NLRP3/IL-1ß) = 0.694 (G). H: Western blotting analysis of NLRP3 (H, first line), TRX (H, second line), TXNIP (H, third line), Caspase-1 (H, fourth line) and IL-1ß expression (H, last line) in the kidneys in the different groups of mice. I: Densitometric analysis of the WB analysis results. Values are the mean ± SE; r: correlation coefficient; *P < 0.05 vs control group; #P < 0.05 vs db/db group. n = 12.

Fig 4: Association of ERBB2 status with TXNRD1 and TXNIP expression. (a) Positive ERBB2 status is associated with significantly higher levels of thioredoxin reductase 1 (TXNDR1) expression. A significant association was obtained in all three individual cohorts and in the combined cohort including 788 patients with node-negative breast cancer. (b) In contrast to TXNDR1, a trend towards lower thioredoxin interacting protein (TXNIP) expression was obtained in ERBB2-status-positive carcinomas. This trend, however, amounted to statistical significance only in the combined cohort.

Fig 5: Induction of ERBB2 (NeuT) by the Tet-on system in MCF-7/NeuT cells influences TXNRD1 and TXNIP. (a) Oncogenic ERBB2 (NeuT) overexpression in MCF-7 cells, induced by exposure to doxycycline, triggers phosphorylation of ERK1/2 and AKT (PKB) as shown by immunoblotting. (b) ERBB2-mediated changes in the transcription of thioredoxin reductase 1 (TXNDR1) and thioredoxin interacting protein (TXNIP) are demonstrated both by Affymetrix Gene Array data and quantitative real-time PCR (qRT-PCR). The gene array graphs display the mean values (± standard deviation) of the Affymetrix probesets. The qRT-PCR graphs show a representative experiment of three with the mean value (± standard deviation) of a triplicate amplification. (c) TXNRD1 and TXNIP protein levels were analyzed by inmmunoblotting. Bands of ~55 kDa for TXNRD1 and ~50 kDa for TXNIP were observed. (d) An increase in accumulation of reactive oxygen species 14 days after onset of ERBB2 (NeuT) expression was shown by detection of malondialdehyde by a thiobarbituric acid reactive substance (TBARS) assay and an N-methyl-2-phenylindole (NMPI) assay. The results represent the mean (± standard deviation) of three experiments, each measured eight times. DETBA, 1,3-diethyl-2-thiobarbituric acid.