Journal: Toxicological Sciences

Article Title: Rotenone Induction of Hydrogen Peroxide Inhibits mTOR-mediated S6K1 and 4E-BP1/eIF4E Pathways, Leading to Neuronal Apoptosis

doi: 10.1093/toxsci/kfu211

Figure Lengend Snippet: Mitochondrial H2O2 elicits apoptosis in rotenone-exposed neuronal cells. The indicated cells were treated with (A and B) rotenone (0.5 and 1 μM) in the presence or absence of TTFA (10 μM) or antimycin A (50 μM) for 24 h, or (C, D, E, and F) pretreated with/without Mito-TEMPO (10 μM) for 1 h and then exposed to rotenone (0.5 and 1 μM) for 24 h, followed by (A, B, and C) H2O2 imaging using a peroxide-selective probe H2DCFDA, (D) cell viability evaluation using the MTS assay, (E) cell apoptosis analysis using DAPI staining, or (F) Western blotting using the indicated antibodies. For (F), the blots were probed for β-tubulin as a loading control. Similar results were observed in at least three independent experiments. For (A), (B), (C), (D), and (E), all data were expressed as means ± SEM (n = 6). ap < .05, difference with control group; b p < .05, difference with 0.5 μM rotenone group; cp < .05, difference with 1 μM rotenone group.

Article Snippet: Mitochondria-targeted antioxidant, Mito-TEMPO and the pan-caspase inhibitor, z-Val-Ala-Asp-CH 2 F (zVAD-fmk) were acquired from ALEXIS Biochemicals Corporation (San Diego, CA).

Techniques: Imaging, MTS Assay, Staining, Western Blot

Journal: Endocrinology

Article Title: Ethyl Pyruvate Preserves IGF-I Sensitivity toward mTOR Substrates and Protein Synthesis in C2C12 Myotubes

doi: 10.1210/en.2010-0248

Figure Lengend Snippet: Selective antioxidants restore IGF-I sensitivity in LPS/IFNγ-treated myotubes. C2C12 myotubes were grown in 10% BCS (control) or treated with either LPS/IFNγ alone or cotreated with LPS/IFNγ and TEMPOL (0.5 mm), mito-TEMPO (0.2 mm), or ethyl pyruvate (EP; 12.5 mm), which were subsequently stimulated with IGF-I during the last 20 min of the experiment. Cell extracts were probed for pS6 (S240/244), total S6, np4E-BP1 (T46), or total 4E-BP1 (A). In a second experiment, myotubes were treated with LPS/IFNγ alone or cotreated with LPS/IFNγ and NAC (6.25 mm), AA (1 mm), or ethyl pyruvate (12.5 mm) in which cells were subsequently stimulated with IGF-I during the last 20 min of the experiment. The resulting blots were probed as described above (B). Blots are representative of at least two similar experiments. The numbers above each lane are the average of duplicate samples and are presented as percent of the control or a fold change from the control.

Article Snippet: Additional experiments tested the efficacy of rapamycin (Biomol, Plymouth Meeting, PA) and an Akt inhibitor (Akt 1/2 inhibitor VIII; Santa Cruz Biotechnology, Santa Cruz, CA) to block IGF-I signaling as well as antioxidants such as TEMPOL (4-hydroxy-TEMPO, Sigma Aldrich), Mito-TEMPO (Biomol), N -acetyl cysteine, and ascorbic acid (all from Sigma Aldrich) to restore IGF-I sensitivity.

Techniques:

Journal: Journal of Neuroinflammation

Article Title: Astrocytes acquire resistance to iron-dependent oxidative stress upon proinflammatory activation

doi: 10.1186/1742-2094-10-130

Figure Lengend Snippet: Role of glutathione and antioxidant molecules against acute iron toxicity in astrocytes. (A) Reduced glutathione (GSH) was measured in lysate of resting (C) or cytokine-activated (CK) astrocytes (average + SEM of three independent experiments). Statistical significance (* p < 0.05) was calculated using unpaired two-tailed Student’s t -test. BSO was used as a control. (B) GSH was measured in resting (C) or activated astrocytes (CK) in basal condition or after 30 min from iron overload. Columns represent the percent of GSH with respect to the control value of resting astrocytes (average +SEM of eight independent experiments). Statistical significance (* p < 0.05; ** p < 0.01) was calculated using one-way ANOVA followed by Bonferroni. (C) A monolayer of resting (BSO) or cytokine-activated (BSO/CK) astrocytes was GSH depleted by BSO, loaded with fura-2 and then challenged by iron. (D) After 90 min from iron overload, cell viability was measured (MTT assay) on astrocytes either in resting condition (C), activated for 24 h with cytokines without (CK) or with (CK/2AAPA) 2AAPA. Each condition was expressed as the percent of viability with respect to the corresponding control without Fe 2+ challenge. Statistical analysis in this and the following panel was performed using one-way ANOVA followed by Bonferroni (*** p < 0.001). (E) After 90 min from acute iron overload, cell viability was measured (MTT assay) on astrocytes in resting condition (C), activated for 24 h with cytokines (CK), or pretreated for 2 h with Mito-TEMPO (MT) or Trolox (* p < 0.05; *** p < 0.001). (F) Astrocytes were plated in culture media containing horse serum from two different companies (BioWhittaker or Invitrogen). Cells were grown for 24 h, then loaded with fura-2 and challenged with iron overload. The traces represent the fluorescence intensity (expressed as RFU) of the astrocytes analyzed by a plate reader.

Article Snippet: In some experiments, a pre-treatment with drugs or pharmacologic agents was needed: (1) L-buthionine-sulfoximine (BSO), an inhibitor of glutathione (GSH) synthesis, 1 mM was added to the cellular medium 24 h before the experiment; (2) Mito-TEMPO [(2-(2,2,6,6-tetramethylpiperidin-1-oxyl-4-ylamino)-2-oxoethyl) triphenylphosphonium chloride monohydrate; ENZO Life Science, Farmingdale, NY, USA] 200 μM, as well as Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) 1 mM were added to cells 2 h before the experiment; (3) the inhibitor of GSH reductase, 2AAPA (R,R’-2-acetylamino-3-[4-(2-acetylamino-2-carboxyethyl-sulfanylthiocarbonylamino)-phenylthiocarbamoylsulfanyl] propionic acid hydrate S,S’-[1,4-Phenylenebis(iminocarbonothioyl)]bis[N-acetyl-L-cysteine], 25 μM, was administered at the beginning of the experiment (after acute iron overload) and left until the end.

Techniques: Two Tailed Test, MTT Assay, Fluorescence

Journal: iScience

Article Title: Low-intensity pulsed ultrasound-generated singlet oxygen induces telomere damage leading to glioma stem cell awakening from quiescence

doi: 10.1016/j.isci.2021.103558

Figure Lengend Snippet:

Article Snippet: Mito-TEMPO , GLPBIO , GC31682.

Techniques: Recombinant, DNA Extraction, TUNEL Assay, Apoptosis Assay