Ogata FT , Batista WL , Sartori A , Gesteira TF , Masutani H , Arai RJ , Yodoi J , Stern A , Monteiro HP .

	Figure 1. HeLa cells submitted to oxidative and nitrosative stress conditions.(A) HeLa cells were treated with increasing concentrations of SNAP (0 - 1.0 mM) during 2 h. After this period medium was replaced for complete medium (MEM supplemented with 10% FBS). Cell viability was determined 24 h later using the MTT assay as described in Materials and Methods. Values are reported in the bar graphs and expressed as means ± S.D. (n = 3, *p < 0.05). (B) Intracellular NO detection in HeLa cells after incubation with 0.5 mM SNAP for 2 h at 37°C. DAF fluorescence is directly related to NO generation by intracellular metabolism of SNAP and was determined by flow cytometry. (C) HeLa cells were treated with increasing concentrations of H2O2 (0 - 1.0 mM) during 2 h. After this period medium was replaced for complete medium (MEM supplemented with 10% FBS). Cell viability was determined 24 h later using the MTT assay as described in Materials and Methods. Values are reported in the bar graphs and expressed as means ± S.D. (n = 3, *p < 0.05). (D) Catalase and Glutathione Peroxidase enzymatic activity assays (described in Materials and Methods) were performed after exposure of HeLa cells to 0.5 mM H2O2 for 2 h at 37°C. (E) HeLa cells were exposed to 0.5 mM SNAP or to 0.5 mM H2O2 for 2 h at 37°C. After cell lysis, 50 μg total protein were subjected to Western blotting with a rabbit polyclonal anti-phospho-Akt antibody and with a mouse monoclonal anti-Akt antibody. Western blot results are representative of three independent experiments. Histogram represents the ratio between the densitometric values of the protein bands corresponding to the phosphorylated form of Akt and of Akt protein of one representative experiment (Lower panel).
	Figure 2. Confocal microscopy and western blot analysis to determine TRX-1 and phospho ERK 1/2 MAP Kinases sub-cellular location in wild-type HeLa cells.(A) Cells were treated with SNAP (0.5 mM), or H2O2 (0.5 mM) for 2 h at 37°C. Detection of TRX-1 and phospho-ERK 1/2 MAP Kinases was performed as described in Materials and Methods. Bar = 20µm. (B) Cells were treated with SNAP (0.5mM) or H2O2 (0.5 mM) for 2 h at 37°C. HeLa cells were pre-treated with 50μM PD98059, a MEK inhibitor, for 30 min and incubated with 0.5mM SNAP or 0.5 mM H2O2 for 2 h at 37°C. Detection of TRX-1 was performed on a fluorescence microscope as described in Materials and Methods. Images shown are representative from three independent experiments. Bar = 20µm. (C) HeLa cells were incubated with SNAP and H2O2 at the indicated concentrations for 2 h and nuclear extracts were subjected to Western blotting with the anti-human TRX-1 mouse monoclonal antibody and with the anti-human phospho-ERK1/2 MAP Kinases rabbit polyclonal antibody. Western blot results are representative of three independent experiments. Purity of nuclear fractions was estimated by LDH detection as described in Materials and Methods. Values are means ± S.D. of triplicate cultures.
	Figure 3. Stress-induced sub-cellular localization and activation of TRX-1 and phospho ERK 1/2 MAP Kinases in HeLa cells expressing PEA-15.(A) HeLa cells and HeLa cells expressing PEA-15 were incubated with SNAP (0.5 mM) or H2O2 (0.5 mM) for 2 h at 37°C. Cells were lysed and phosphorylation of the ERK1/2 MAP kinases was examined by western blotting. Western blot results are representative of four independent experiments. Histogram represents the ratio between the densitometric values of the protein bands corresponding to the phosphorylated form and of the total ERK 1/2 MAP kinases (Lower panel). (B) HeLa cells expressing PEA-15, were treated with SNAP (0.5 mM), or H2O2 (0.5 mM) for 2 h at 37°C. Sub-cellular localization of TRX-1 and phospho-ERK 1/2 MAP Kinases was performed as described in Materials and Methods. Bar = 20µm. (C) HeLa cells and HeLa cells expressing PEA-15 were incubated with SNAP (0.5 mM) or H2O2 (0.5 mM) for 2 h at 37°C. Nuclear extracts were subjected to Western blotting with the anti-human TRX-1 mouse monoclonal antibody. Western blot results are representative of three independent experiments.
	Figure 5. SNAP and H2O2 -modulated TXNIP mRNA and protein levels in wild-type Hela cells and in HeLa cells expressing PEA-15.(A) Relative levels of TXNIP mRNA determined by quantitative real-time PCR in HeLa cells wild-type, in HeLa cells expressing PEA-15, and in HeLa cells wild-type pre-treated with 50μM of PD98059 for 30 min as indicated (see Materials and Methods for details). All cell lines were treated with 0.5mM SNAP or 0.5mM H2O2 for 2 h at 37°C. Values are reported in the bar graphs and expressed as means ± S.D. (n = 3, *p < 0.05; **p < 0.01). (B) wild-type HeLa cells and HeLa cells expressing PEA-15 were incubated with SNAP (0.5 mM) or H2O2 (0.5 mM) for 2 h at 37°C. Cells were lysed and expression levels of TXNIP were examined by western blotting. Western blot results are representative of four independent experiments. Histogram represents the ratio between the densitometric values of the protein bands corresponding to TXNIP and of β-actin (Lower panel).
	Figure 6. TXNIP silencing and TRX-1 intracellular localization.(A) Western blot analysis to determine the effects of mRNA TXNIP knockdown on TXNIP protein levels in HeLa cells permanently transfected with a shRNA for TXNIP. The blot presented is representative of three independent experiments. (B) Indirect immunofluorescence to determine TRX-1 sub-cellular localization in HeLa cells permanently transfected with a shRNA for TXNIP. Images shown are representative from three independent experiments. Blue arrows indicate TRX-1 nuclear accumulation. Bar = 20µm.
	Figure 7. Confocal microscopy based analysis to determine a time-dependent sub-cellular localization of TRX-1 in HeLa cells.(A) Wild-type HeLa cells, HeLa cells expressing PEA-15, and HeLa cells expressing TXNIP were plated two days prior to experiment. Transient transfections with pEGFP-C1_TRX-1 were performed on day one. On day two, cells were assayed for TRX-1 sub-cellular localization. Cells were kept in a heated stage chamber (37°C) of the confocal microscope, and a first scanning was recorded to serve as control. After that, media was replaced and H2O2 was added to a final concentration of 0.5 mM. Subsequent scanning were performed at every 15 min up to 2 h. Bar = 20µm. (B) Cells were transfected with pEGFP-C1-TRX-1, treated with SNAP (0.5 mM) for 2 h at 37°C and submitted to indirect immunofluorescence for GFP, to show the localization of TRX-1 in HeLa cells. Red arrows indicate TRX-1 nuclear accumulation.
	Figure 8. Determination of sub-cellular compartmentalization of TRX-1 in HeLa cells transiently over-expressing PEA-15; TXNIP; TRX-1 and exposed to 0.5 mM H2O2.HeLa wild-type cells were seeded in complete medium and 24 h later were transiently transfected with the following plasmids pcDNA3_PEA-15, pcDNA3_TXNIP, and pEGFP-C1_TRX-1. After 16 h, transiently transfected cultures were divided to perform either western blot analysis, to confirm over expression of designated inserts, or to perform immunofluorescence-based analysis to determine TRX-1 sub cellular localization after exposure to 0.5 mM H2O2. (A) HeLa cells transfected with pcDNA3_PEA-15. Left panel: western blot analysis of PEA-15 expression. Right panel: Immunofluorescence image of TRX-1 cellular location. (B) HeLa cells transfected with pcDNA3_TXNIP. Left panel: western blot analysis of TXNIP expression. Right panel: Immunofluorescence image of TRX-1 cellular location (C) HeLa cells transfected with pEGFP-C1_TRX-. Left panel: western blot analysis of TRX-1 expression. Right panel: Immunofluorescence image of TRX-1 cellular location. Red arrows indicate TRX-1 nuclear accumulation.
	Figure 9. Schematic representation of TRX-1 and the ERK1/2 MAP kinases nuclear translocation stimulated by nitrosative/oxidative stress conditions.(A) The Ras-Raf-MEK-ERK 1/2 signaling pathway is activated in HeLa wild-type cells after exposure to 0.5 mM SNAP or 0.5 mM H2O2 (1). Under these conditions expression of TXNIP is down-regulated at mRNA and protein levels (2) and TRX-1 and the ERK 1/2 MAP kinases, independently of each other, migrate to the nuclear compartment (3). (B) Nuclear migration of TRX-1 and the ERK 1/2 MAP kinases is prevented in three situations: HeLa cells pre-incubated with the MEK inhibitor PD98059 (4), HeLa cells over-expressing the cytoplasmic anchor of ERK 1/2 MAP kinases – PEA-15 (5), HeLa cells over-expressing the physiological inhibitor of TRX-1, TXNIP (6).

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