Fig 1: Response of the thioredoxin system to Sorafenib and siRNATrx1 treatments. Changes in the levels of three components of the thioredoxin system, (A) Trx1, (B) TrxR1 and (C) TXNIP were determined in HepG2, SNU423 and SNU475 cells by Western blotting with their specific antibodies on treatment with Sorafenib or siRNATrx1 independently or combined under the conditions described in M&M. Densitometric data normalized for ß-actin are shown; samples from 3 different experiments (N = 3) for each cell line were run in the same gel; protein levels between cells cannot be compared since each set of 3 replicas for each cell line was developed on a different WB membrane; for a comparison of Trx1, TrxR1 between cell lines, refer to quantitative proteomic data in Figure 1; a composition of representative blots is shown below each graph. p values 0.0332 (*), 0.0021 (**), 0.0002 (***), <0.0001 (****), see Materials & Methods for statistical details.
Fig 2: Epithelial–mesenchymal transition (EMT) markers and thioredoxin system in three hepatocarcinoma (HCC) cells lines. Data for vimentin, alpha-fetoprotein, CD44 antigen, Trx1, and TrxR1 were retrieved from the quantitative proteomic analysis of HepG2, SNU423, and SNU475 cells (Supplementary File S1). The scale in the vertical axis is the relative intensity from the LC–MS/MS quantitative analysis and varies between proteins; the maximum value for each protein ranges from 3.25e + 007 for alpha-fetoprotein in HepG2 cells to 8.97e + 008 for Trx1 in SNU475 cells. (N = 3, individual values are shown).
Fig 3: Structural comparison and sequence motif analysis of redox cysteine position in Nrf2 and Keap1 knockouts. (A) Superimposition of 3-D structure of peptides harboring redox cysteine (highlighted in ball and stick model). (B) Primary sequence motifs for modified cysteine residues. The central ‘‘C’’ represents the redox cysteine residue. The size of the letter represents the probability of the residue. (C) Western blot analysis of Sdhd (left panels) and Txnrd1 (right panels) of muscle in Nrf2-WT, Nrf2-KO, Keap1-WT, and Keap1-KO mouse models. Up panels: raw data of Western blots; Bottom panels: group data showing as mean ± SD; n = 4 for each group.
Fig 4: Immunostaining of proteins ASK1, RNR, and TrxR1 in C6, RC6, U87, and U87-TxR cell lines. Changes in expression of proteins in cells treated with 2 µM 5 and 8 µM 6 are compared to untreated control (set at 100%, red line). All results represent mean values ± SD, obtained from three independent experiments (n = 3). p < 0.05 (*), p < 0.01 (**), and p < 0.0001 (****) indicate significantly different levels of expression in cells treated with compound 5 in comparison with untreated control. p < 0.05 (#) and p < 0.001 (###) indicate statistical significance between untreated control and cells treated with 6.
Fig 5: Quantitative real-time PCR analysis of changes in antioxidative enzymes expression in C6, RC6, U87, and U87-TxR cell lines, induced by 2 μM 5 and 8 μM 6. The mRNA expression of Trx1, TrxR1, GPx1, GPx4, GSTπ, GR, MnSOD, and CAT was normalized to ACTB as internal control. All results represent mean values ± SD, obtained from three independent experiments (n = 3). p < 0.01 (**), p < 0.001 (***), and p < 0.0001 (****) indicate significantly different level of expression in cells treated with 5 in comparison with untreated control. p < 0.05 (#), p < 0.01 (##), and p < 0.0001 (####) indicate statistical significance between untreated control and cells treated with 6.
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