Tert butylhydroperoxide t booh

ISL was provided by Shanghai Yuanye Biotechnology Co., Ltd. (Shanghai, China). (−)-Nicotine hydrogen tartrate, tert-butyl hydroperoxide (t-BOOH), and NMDA were purchased from Sigma-Aldrich (St. Louis, MO, USA). Primary antibodies against total extracellular regulated protein kinases 1/2 (Erk1/2), phospho (P)-Erk1/2, and β-actin were obtained from Abcam (Cambridge, UK), and horseradish peroxidase-conjugated secondary antibody was purchased from Cell Signaling Technology (Beverly, MA, USA).

Cortical and dopaminergic mid-brain neurons were isolated from E18.5 Sprague-Dawley rats and plated on poly-L-lysine coated plates in complete Neurobasal/B27 (Invitrogen). Mid-brain neurons were cultured with GDNF (10 ng/ml), BDNF (10 ng/ml) and dbcAMP (0.5 mM) for 6 days for dopaminergic neuron maturation. Cells then were switched to Neurobasal/B27 media without antioxidants, supplemented with GRP, GDA^BMP or GDA^CNTF conditioned medium (CM) for 24 h. In some groups, cells were further challenged by addition of tert-butyl hydroperoxide (tBOOH,1 μM, Sigma, St. Louis, MO) or 6-hydroxy-dopamine (6-OHDA, 10 μM, Sigma, St. Louis, MO). Cell survival was determined by co-labeling cultures with Tuj1 and anti-Tyrosine hydroxylase antibody (Secondary antibodies: anti-mouse IgG1-Alexa 488 and anti-rabbit Ig Alexa 568, Invitrogen). TujI⁺ or TujI⁺/TH⁺ neurons were counted using a Celigo® adherent cell cytometer (Brooks, Chelmsford, MA). Results are presented as mean ± s.d. (n = 3). Statistical analysis was performed using One-Way ANOVA, pairwise Bonferroni Multiple Comparison Test (*P < 0.05; Prism 5, Graphpad, La Jolla, CA).

pCH9/3091 was obtained from Lin Lan (The Third Military Medical University, Chongqing, China). pCH9 was constructed by digesting the HBV genome in the pCH9/3091 and ligating with T4 DNA ligase (Takara, Kusatsu, Shiga, Japan). The MUT HBV plasmid was constructed by site-directed mutagenesis of pCH9/3091 (as the wild-type HBV, WT HBV) via introduction of a stop codon at the beginning of the HBx gene. Site-directed mutagenesis was carried out by PCR amplification of the WT HBV. The primer carried a C-to-T mutation at nt 1397. This mutation results in a stop codon mutation in the HBx gene (codon 8) without affecting the polymerase gene product. pcDNA3.1-Flag-SIRT3 was obtained from ADDGENE (Cambridge, MA, USA). SiRNA targeted SIRT3 was obtained from Invitrogen (Carslbad, CA, USA). Rabbit anti-SIRT3 monoclonal antibody, anti-γ-H₂AX monoclonal antibody and anti-PRDX1 monoclonal antibody were obtained from Cell Signaling Technology (CST, Danvers, MA, USA). Rabbit anti-Flag monoclonal antibody was obtained from Sigma (St Louis, MO, USA). Rabbit anti-β-actin monoclonal antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Tetracycline, tert-butyl hydroperoxide (tBOOH) and H₂O₂ were obtained from Sigma. NAC (S0077) was obtained from Beyotime (Haimen, Jiangsu, China).

To evaluate the protective effect of preparations against oxidative stress, cells were pre-incubated with IC₀ (concentration at which the viability amounted to 100%) of extracts for 24 h. Then, to induce oxidative stress conditions, 500 μM tert-butylhydroperoxide (t-BOOH) (Sigma-Aldrich, St. Louis, MO, USA) was added for 2 h and viability of cells was measured.
To determine the effect of extracts on the intracellular generation of ROS, cells were incubated with extracts for 24 h and afterwards—loaded with DCFH-DA (Sigma-Aldrich, St. Louis, MO, USA) dye at a final concentration of 10 μM for 30 min. Fluorescent signal was analyzed at F485/530 nm.

To determine the activity of the antioxidant enzymes GPx and GR, pancreas samples (400 μg of protein) were mixed in Tris buffer 0.25 M, sucrose 0.2 M, and DTT buffer 5 mM at pH 7.4, and then centrifuged at 3000× g for 15 min. GPx activity is based on the oxidation of GSH by GPx (Sigma-Aldrich) using tert-butylhydroperoxide (t-BOOH, Sigma-Aldrich, Madrid, Spain) as a substrate, coupled to the disappearance of nicotine adenine dinucleotide phosphate reduced salt (NADPH, Sigma Aldrich, Madrid, Spain) by GR. GR activity was analyzed by monitoring the oxidation of NADPH, which is used in the reduction of GSSG [23 (link)].

R. sphaeroides strains (Table C in S1 File) were cultivated in malate minimal salt medium [14 (link)] at 32°C in the dark. Conditions of semi-aerobic (~25 μM dissolved oxygen) and aerobic growth (~180 μM dissolved oxygen) were accomplished as recently described [28 (link)]. Iron-limiting conditions were achieved as described in [15 (link)]. Stress experiments were conducted in exponential growth phase (OD₆₆₀ of 0.4). Singlet oxygen (¹O₂) was generated by the addition of 0.2 μM methylene blue (Sigma-Aldrich) in the presence of high light intensities (800 W m^-2 white light). Hydrogen peroxide (H₂O₂; Roth) and tert-butyl hydroperoxide (tBOOH; Sigma-Aldrich) were added in final concentrations of 1 mM and 300 μM, respectively. 250 μM of paraquat (PQ) were used for the generation of superoxide radicals (O₂^‒). Samples taken at the indicated time-points were rapidly cooled on ice and harvested by centrifugation at 10,000 x g in a chilled centrifuge.

The different concentrations of H₂O₂ (1.0 mM, 1.5 mM, and 2.0 mM) and tert-Butyl hydroperoxide (t-BOOH) (0.2 mM, 0.3 mM and 0.4 mM) (Sigma-Aldrich) in the CM solid medium were used to detect the sensitivity of WT, VdTrx1 mutant, and VdTrx1 complemented strains to oxidative stress. Otherwise, 1 M sorbitol (Solarbio) and 200 μg/ml Congo red (Sigma-Aldrich) were added to the CM medium to assay osmotic stress and cell-wall integrity.
To measure the penetration ability of the wild type, the VdTrx1 mutant, and the VdTrx1 complemented strains, 5 μl of the spore suspensions of each, with concentrations of 5 × 10⁶ conidia/ml, was cultured on MM plate covered with sterilized cellophane membrane and incubated at 25°C. The cellophane membranes were removed after 4 days. The plates were further cultured for 3 days at 25°C to observe the hyphae that had passed through the membrane forming colonies.