Journal: Redox Biology

Article Title: Tert-butyl hydroperoxide (t-BHP) induced apoptosis and necroptosis in endothelial cells: Roles of NOX4 and mitochondrion

doi: 10.1016/j.redox.2016.12.036

Figure Lengend Snippet: T-BHP H induced endothelial necroptosis through RIP1-RIP3-MLKL Endothelial cells were treated with t-BHP H for 1 h with or without Nec-1 (1 μM) pretreatment for 1 h, the interaction of RIP1 and RIP3, and the phosphorylation of MLKL were detected by immunoprecipitation (A) and Western blotting (B), respectively. RIP1, RIP3, and MLKL were silenced by siRNAs (C), the cell viability in response to t-BHP H for 1 h was detected by LDH assay kit (D). The results were expressed as mean±SD of three independent experiments. Cont, control group.

Article Snippet: After determination of the protein concentrations, the cell extracts were incubated with anti-RIP1 antibody (2 μg) for 2 h at 4 °C and followed by incubation with 20 μL of proteinA/G plus-agarose beads overnight.

Techniques: Phospho-proteomics, Immunoprecipitation, Western Blot, Lactate Dehydrogenase Assay, Control

Journal: Redox Biology

Article Title: Tert-butyl hydroperoxide (t-BHP) induced apoptosis and necroptosis in endothelial cells: Roles of NOX4 and mitochondrion

doi: 10.1016/j.redox.2016.12.036

Figure Lengend Snippet: Mitochondrial ROS was required for activation of t-BHP H -induced endothelial necroptosis. Endothelial cells were treated with t-BHP (50–500 μM) 1 h with or without Rot (20 μM), TTFA (10 μM), AA (5 μM) pretreatment for 1 h, ROS generation and LDH release were detected by flow cytometer (A and B) and LDH assay kit (C). After transfected with RIP1, RIP3, or MLKL siRNAs, cells were treated with t-BHP H for 15 min. The MMP and mitochondrial ROS generation were detected with JC-1 staining (D) and MitoSOX by fluorescence microscopy (E) and detected by FACScan flow cytometer (F). Cells were treated with t-BHP H for 1 h with or without Rot (20 μM), TTFA (10 μM), AA (5 μM) pretreatment for 1 h, RIP1-RIP3 interaction and MLKL phosphorylation were detected by immunoprecipitation (G) and Western blotting (H). The results were expressed as mean±SD of three independent experiments. AA, antimycin A; ALL, allopurinol; Cont, control group; DPI, diphenyleneiodonium; LDH, lactate dehydrogenase; MMP, mitochondrial membrane potential; ROS, reactive oxygen species; Rot, rotenone; TTFA, 2-thenoyltrifluoroacetone.

Article Snippet: After determination of the protein concentrations, the cell extracts were incubated with anti-RIP1 antibody (2 μg) for 2 h at 4 °C and followed by incubation with 20 μL of proteinA/G plus-agarose beads overnight.

Techniques: Activation Assay, Flow Cytometry, Lactate Dehydrogenase Assay, Transfection, Staining, Fluorescence, Microscopy, Phospho-proteomics, Immunoprecipitation, Western Blot, Control, Membrane

Journal: Redox Biology

Article Title: Tert-butyl hydroperoxide (t-BHP) induced apoptosis and necroptosis in endothelial cells: Roles of NOX4 and mitochondrion

doi: 10.1016/j.redox.2016.12.036

Figure Lengend Snippet: P38MAPK was involved in t-BHP H -induced endothelial necroptosis Endothelial cells were treated with t-BHP H for 15 min with or without Nec-1 (1 μM) pretreatment for 1 h, expressions of total and phosphorylated JNK1/2, p38MAPK and ERK1/2 were detected by Western blotting (A). Endothelial cells transfected with siRNA against RIP1 or MLKL were treated with t-BHP H for 15 min, the expression of phosphorylated p38MAPK was analyzed by Western blotting (B). Endothelial cells were treated t-BHP H for 1 h with or without SB203580 (1 μM) pretreatment for 1 h, the cell viability and the LDH release were examined by MTT assay (C) and LDH assay kit (D), respectively. (E) Hypothetical signal pathway of t-BHP-induced endothelial apoptosis and necroptosis in endothelial cells. These results were expressed as mean±SD of three independent experiments. LDH, lactate dehydrogenase;Nec-1, necrostatin-1.

Article Snippet: After determination of the protein concentrations, the cell extracts were incubated with anti-RIP1 antibody (2 μg) for 2 h at 4 °C and followed by incubation with 20 μL of proteinA/G plus-agarose beads overnight.

Techniques: Western Blot, Transfection, Expressing, MTT Assay, Lactate Dehydrogenase Assay

Journal: Redox Biology

Article Title: Tert-butyl hydroperoxide (t-BHP) induced apoptosis and necroptosis in endothelial cells: Roles of NOX4 and mitochondrion

doi: 10.1016/j.redox.2016.12.036

Figure Lengend Snippet:

Article Snippet: After determination of the protein concentrations, the cell extracts were incubated with anti-RIP1 antibody (2 μg) for 2 h at 4 °C and followed by incubation with 20 μL of proteinA/G plus-agarose beads overnight.

Techniques: