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: 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: American Journal of Physiology - Cell Physiology

Article Title: ROS-induced ROS release orchestrated by Nox4, Nox2, and mitochondria in VEGF signaling and angiogenesis

doi: 10.1152/ajpcell.00346.2016

Figure Lengend Snippet: Nox4 overexpression- or H2O2 -induced mitochondrial ROS production is inhibited by Nox2 knockdown in ECs. A and B: HUVECs were transfected with siCtrl or siNox2 for 24 h, and then infected with adenovirus expressing Nox4 (Ad.Nox4) or Ad.control (Ad.Ctrl) for 24 h. Cells were used to measure DCF fluorescence (A) or MitoSox fluorescence (B). C: HUVECs transfected with siCtrl or siNox2 or siNox4 or treated with Mito-TEMPO (50 μM) were stimulated with H2O2 (500 μM) for 20 min and then used to measure MitoSox fluorescence. Graph represents averaged MitoSox fluorescence intensity expressed as fold increase over the basal (n = 3, *P < 0.05). D: scheme showing that Nox4-derived H2O2 activates Nox2 that produce O2−/H2O2, which in turn promotes mitochondrial ROS (mtROS) production in ECs.

Article Snippet: We confirmed that MitoSox fluorescence was abolished by mitochondria-targeted SOD mimetic, Mito-TEMPO (Enzo).

Techniques: Over Expression, Transfection, Infection, Expressing, Fluorescence, Derivative Assay

Journal: American Journal of Physiology - Cell Physiology

Article Title: ROS-induced ROS release orchestrated by Nox4, Nox2, and mitochondria in VEGF signaling and angiogenesis

doi: 10.1152/ajpcell.00346.2016

Figure Lengend Snippet: VEGF-induced pSer36-p66Shc is required for mitochondrial ROS production in ECs. A and B: HUVECs were infected with Ad.p66Shc-S36A or Ad.control (Ad.Ctrl) (A) or treated with Mito-TEMPO (50 μM) for 24 h (B). Cells were stimulated with VEGF (20 ng/ml) for 30 min, and then used to measure MitoSox fluorescence. Graph represents the averaged MitoSox fluorescence intensity. C: HUVECs were infected with Ad.Ctrl, Ad.Mito-catalase, or Ad.p66Shc-S36A for 48 h. Cells were stimulated with VEGF (20 ng/ml) for 15 min and then used to measure MitoTracker CM-H2TMRos fluorescence imaged by confocal microscopy. Graph represents averaged fluorescence intensity expressed as fold increase over the basal. (n = 3, *P < 0.05).

Article Snippet: We confirmed that MitoSox fluorescence was abolished by mitochondria-targeted SOD mimetic, Mito-TEMPO (Enzo).

Techniques: Infection, Fluorescence, Confocal Microscopy

Journal: American Journal of Physiology - Cell Physiology

Article Title: ROS-induced ROS release orchestrated by Nox4, Nox2, and mitochondria in VEGF signaling and angiogenesis

doi: 10.1152/ajpcell.00346.2016

Figure Lengend Snippet: VEGF-induced pSer36-p66Shc is required for VEGFR2 activation and angiogenic responses in ECs. A and B: HUVECs were infected with Ad.p66Shc-S36A or Ad.control (Ad.Ctrl) (A) or treated with Mito-TEMPO (50 μM) for 24 h (B). Cells were stimulated with VEGF for indicated times, and lysates were IB with VEGFR2-pY1175 or total VEGFR2 Abs (A); or IP with anti-Shc Ab, followed by IB with anti-pS36-p66Shc or Shc Abs (A and B), Graph represents p-VEGFR2 per total VEGFR2 expressed as fold change over the basal (A) or pS36-p66shc per total p66shc expressed as fold change over the basal (B) (n = 3, *P < 0.05). C: cell migration stimulated with VEGF for 6 h was measured using modified Boyden chamber method (left). For cell proliferation, cells were cultured in 0.5% FBS containing medium with or without VEGF (20 ng/ml) for 48 h and the cell number was counted (right) (n = 3, *P < 0.05).

Article Snippet: We confirmed that MitoSox fluorescence was abolished by mitochondria-targeted SOD mimetic, Mito-TEMPO (Enzo).

Techniques: Activation Assay, Infection, Migration, Modification, Cell Culture