Journal: PLoS ONE

Article Title: Desipramine Protects Neuronal Cell Death and Induces Heme Oxygenase-1 Expression in Mes23.5 Dopaminergic Neurons

doi: 10.1371/journal.pone.0050138

Figure Lengend Snippet: Cells were pretreated with various of MAP kinase inhibitors SB203580 (10 µM), PD98059 (20 µM) or SP600125 (10 µM) for 30 min followed by stimulation with desipramine (20 µM; A) or fluoxetine (20 µM; D) for another 24 h. Whole cell lysates were subjected to Western blot for detection of HO-1 expression. Cells were incubated with desipramine or fluoxetine for the indicated time periods, and cell lysates were subjected to immunoblots with antibodies against phospho-JNK (B and C) and phospho-ERK (D and E). Results are the representatives of three or four independent experiments.

Article Snippet: SP600125 was obtained from Tocris Bioscience (Ellisville, MO).

Techniques: Western Blot, Expressing, Incubation

Journal: PLoS ONE

Article Title: Desipramine Protects Neuronal Cell Death and Induces Heme Oxygenase-1 Expression in Mes23.5 Dopaminergic Neurons

doi: 10.1371/journal.pone.0050138

Figure Lengend Snippet: (A) Cells were treated with desipramine (20 µM) for indicated time periods (60 or 120 min) and nuclear extracts were collected, and the binding activity of Nrf2 to Nrf2-DNA binding element was examined by EMSA analysis. The DNA binding activity of Nrf2 is significantly different between desipramine treatment group and control group (one-way ANOVA followed by Bonferroni’s post hoc test). Cells were pretreated with PD98059 or SP600125 with desipramine (20 µM), and nuclear extracts were examined by EMSA analysis. Lane 1 was loaded without nuclear extracts (probe only). Results are expressed as the means ± S.E.M. from three independent experiments. *, p <0.05 as compared with the vehicle control group. #, p<0.05 as compared with the desipramine treatment group. (B) Cells were transfected with Control siRNA (100 nM) or Nrf2 siRNA (50 and 100 nM) for 24 h followed by stimulation with desipramine (20 µM) for another 24 h, and the protein levels of Nrf2 and HO-1 were determined by Western blot. The HO-1 expression is significantly different between Nrf2 siRNA group and control siRNA group (one-way ANOVA followed by Bonferroni’s post hoc test). Results are expressed as the means ± S.E.M. from three independent experiments. *, p <0.05 as compared with the control group. #, p<0.05 as compared with the desipramine treatment alone group.

Article Snippet: SP600125 was obtained from Tocris Bioscience (Ellisville, MO).

Techniques: Binding Assay, Activity Assay, Control, Transfection, Western Blot, Expressing

Journal: Cell reports

Article Title: JNK Phosphorylates SIRT6 to Stimulate DNA Double-Strand Break Repair in Response to Oxidative Stress by Recruiting PARP1 to DNA Breaks

doi: 10.1016/j.celrep.2016.08.006

Figure Lengend Snippet: (A) CoIP reveals that SIRT6 interacts with JNK in HCA2-hTERT cells only when the cells have been exposed to oxidative stress (1 mM paraquat for 16hr). The experiment was repeated at least four times. (B) In vitro phosphorylation assay demonstrating that JNK can phosphorylate SIRT6 in vitro. Anisomysin-activated JNK, purified from HEK293 cells, was incubated with BSA and bacterially purified SIRT6 in the presence of 32 P-ATP and a kinase reaction buffer. SIRT6 specifically incorporated the radiolabel in these reactions, indicating that it was phosphorylated by JNK. The experiment was repeated three times and a representative gel is shown. See also . (C) SIRT6 plasmids encoding mutations at the indicated putative phosphorylation sites were overexpressed in HCA2-hTERT-NHEJ cells to measure their ability to stimulate NHEJ. SIRT6 T294A, S303A, S330A, and S338A all stimulated NHEJ similarly to WT SIRT6. SIRT6 S10A, however, failed to stimulate NHEJ in response to stress. Expression of an SIRT6 plasmid encoding an S10E phospho-mimetic mutation was able to powerfully stimulate NHEJ in the absence of oxidative stress. The effect of S10E mutation on DNA repair was resistant to JNK inhibition with SP600125. Error bars indicate SD (n = 4). Immunoblot (above) demonstrates that all of the indicated SIRT6 vectors were expressed stably and at comparable levels (*p < 0.05 and **p < 0.01). (D) NHEJ reporter construct was integrated into SIRT6 −/− MEF to measure the SIRT6 S10A and S10E activity in DNA repair in the absence of endogenous SIRT6. Cells expressing SIRT6 S10A mutant showed no stimulation of NHEJ repair in response to paraquat-induced oxidative stress. SIRT6 S10E phospho-mimetic mutant stimulated NHEJ under basal conditions, which could be further stimulated by stress; however, this additional stimulation of NHEJ was not affected by JNK inhibitor SP600125. Error bars indicate SD (n = 3; *p < 0.05 and **p < 0.01). (E) In vitro phosphorylation assay demonstrates that, while JNK can phosphorylate WT SIRT6, it cannot phosphorylate SIRT6 S10A. Anisomysin-activated JNK, purified fromHEK293 cells, was incubated with BSA and bacterially purified WT SIRT6 of SIRT6 S10A in the presence of 32 P-ATP and a kinase reaction buffer. The experiment was repeated three times and a representative gel is shown. (F) SIRT6 is phosphorylated on S10 in vivo after oxidative stress and the phosphorylation is diminished by JNK inhibitor (SP600125). Custom rabbit polyclonal antibodies (Rb5159 and Rb5160) were generated by immunizing rabbits with YAAGL pS PYADKGKC peptide (see for antibody specificity assays). The hTERT-immortalized human fibroblasts HCA2 were transfected with WT SIRT6-expressing plasmid, then treated with paraquat and/or JNK inhibitor and SIRT6 S10-P, and total SIRT6 levels were assessed by western blot (Rb5159 is pictured; both antibodies gave comparable results). To further confirm the specificity of S10-P antibodies, replicate samples were run and treated with Lambda Protein Phosphatase (LPP) for 1 hr, prior to antibody staining. The experiment was repeated three times and a representative blot is shown.

Article Snippet: Purified, anisomycin-activated JNK (1 µg, Signal Chem, M34-10G) was incubated with 5 µg bacterially purified recombinant SIRT6 for 2 hr in Kinase Reaction Buffers (Signal Chem, K23-09-05 and K21-09-05) and ATP or [γ- 32 P]-ATP.

Techniques: In Vitro, Phosphorylation Assay, Purification, Incubation, Expressing, Plasmid Preparation, Mutagenesis, Inhibition, Western Blot, Stable Transfection, Construct, Activity Assay, In Vivo, Generated, Transfection, Staining