adeno associated virus type 1 expressing faf1  (Vector Biolabs)

Journal: Cell Death and Differentiation

Article Title: FAF1 mediates regulated necrosis through PARP1 activation upon oxidative stress leading to dopaminergic neurodegeneration

doi: 10.1038/cdd.2016.99

Figure Lengend Snippet: FAF1 is required for PARP1-dependent necrosis after H2O2 treatment. (a) Left panel: WT MEFs were transfected with the vector control (VC) or Flag-FAF1 plasmids. At 36 h after transfection, cells were pretreated with vehicle (DMSO), zVAD-fmk (100 μM) or DPQ (30 μM) for 1 h and were then treated with 500 μM H2O2 for 6 h in presence of individual compounds. Cell death was determined on the basis of LDH release (n=3). Right panel: representative immunoblots show the levels of FAF1, Flag and β-actin expression. (b) Immunoblot analysis of FAF1 expression in immortalized Faf1+/+ and Faf1gt/gt MEFs. β-actin expression was used as an endogenous control. (c) Faf1+/+ and Faf1gt/gt MEFs were treated with the indicated concentrations of H2O2 for 6 h. Cell death was determined on the basis of LDH release (n=3). (d) Faf1+/+ and Faf1gt/gt MEFs were treated with 500 μM H2O2 for the indicated times. Cell death was determined on the basis of LDH release (n=3). (e) Left panel: Faf1gt/gt MEFs were transfected with the VC or Flag-FAF1 plasmids. Thirty-six hours after transfection, the cells were treated with 500 μM H2O2 for 6 h. Cell death was determined on the basis of LDH release (n=3). Right panel: representative immunoblots show the levels of FAF1, Flag and β-actin expression. Data (a, c–e) are expressed as the mean±S.E.M. from three independent experiments. Statistical comparisons were evaluated by ANOVA test followed by Tukey HSD (a, c–e) post hoc analysis. ***P<0.001, **P<0.01 and *P<0.05

Article Snippet: In brief, the adeno-associated virus type 1 expressing FAF1 (AAV1-FAF1) was purchased from Vector Biolabs (Malvern, PA, USA).

Techniques: Transfection, Plasmid Preparation, Control, Western Blot, Expressing

Journal: Cell Death and Differentiation

Article Title: FAF1 mediates regulated necrosis through PARP1 activation upon oxidative stress leading to dopaminergic neurodegeneration

doi: 10.1038/cdd.2016.99

Figure Lengend Snippet: FAF1 translocates to the nucleus and interacts with PARP1 during oxidative stress. (a) WT MEFs were treated with 500 μM H2O2 for the indicated times and were then fractionated into cytoplasmic and nuclear fractions. The fractions were analyzed by immunoblotting with anti-FAF1, anti-PARP1 (nuclear marker) and anti-β-actin (cytoplasmic marker) antibodies. (b) WT MEFs were treated with 500 μM H2O2 for the indicated times and were then immunostained with the anti-FAF1 antibody. The nuclei were stained using propidium iodide (PI) and analyzed by confocal microscopy. (c) WT MEFs were transfected with the indicated combination of V5-PARP1 and Flag-FAF1 plasmids. At 36 h after transfection, the cells were untreated or treated with 500 μM H2O2 for 30 min. The cell lysates were immunoprecipitated with anti-Flag antibody, followed by immunoblotting with the indicated antibodies. (d) WT MEFs were treated with 500 μM H2O2 for the indicated times. The cell lysates then were immunoprecipitated with the anti-FAF1 antibody. The interactions were determined by immunoblotting with the indicated antibodies

Article Snippet: In brief, the adeno-associated virus type 1 expressing FAF1 (AAV1-FAF1) was purchased from Vector Biolabs (Malvern, PA, USA).

Techniques: Western Blot, Marker, Staining, Confocal Microscopy, Transfection, Immunoprecipitation

Journal: Cell Death and Differentiation

Article Title: FAF1 mediates regulated necrosis through PARP1 activation upon oxidative stress leading to dopaminergic neurodegeneration

doi: 10.1038/cdd.2016.99

Figure Lengend Snippet: FAF1 promotes the catalytic activity of PARP1 during oxidative stress. (a) Left panel: WT MEFs were transfected with the indicated concentration of Flag-FAF1 plasmids. At 36 h after transfection, the cells were treated with 500 μM H2O2 for 30 min. The cell lysates were immunoblotted with the indicated antibodies. Right panel: the graph shows the result of densitometric analysis of PAR immunoblots (n=3). (b) Left panel: WT MEFs were transfected with the VC or Flag-FAF1 plasmids. At 36 h after transfection, the cells were treated with 500 μM H2O2 for the indicated times. The cell lysates were immunoblotted with the indicated antibodies. Right panel: the graph shows the result of densitometric analysis of PAR immunoblots (n=3). (c) Left panel: Faf1+/+ and Faf1gt/gt MEFs were treated with 500 μM H2O2 for the indicated times. The cell lysates were subjected to immunoblot analysis with the indicated antibodies. Right panel: the graph shows the result of densitometric analysis of PAR immunoblots (n=3). (d) Faf1+/+ and Faf1gt/gt MEFs were treated with 500 μM H2O2 for 30 min and were then immunostained with the anti-PAR antibody. The nuclei were stained using PI and the cells were analyzed by confocal microscopy. (e) Left panel: Faf1gt/gt MEFs were transfected with the VC or Flag-FAF1 plasmids. Thirty-six hours after transfection, the cells were treated with 500 μM H2O2 for the indicated times. The cell lysates were subjected to immunoblot analysis with the indicated antibodies. Right panel: the graph shows the result of densitometric analysis of PAR immunoblot (n=3). (f) Left panel: GST-FAF1 or GST was incubated with recombinant PARP1 (1 unit), β-NAD+ (100 μM) and damaged DNA for 10 min at room temperature. After the in vitro poly(ADP-ribosyl)ation reactions, the samples were subjected to immunoblot analysis. Right panel: the graph shows the results of densitometric analysis of PAR immunoblots (n=3). Quantified data (a–c, e, f) are expressed as the mean±S.E.M. from three independent experiments. Statistical comparisons were evaluated by ANOVA test followed by Tukey HSD (a–c, e, f) post hoc analysis. **P<0.01 and *P<0.05

Article Snippet: In brief, the adeno-associated virus type 1 expressing FAF1 (AAV1-FAF1) was purchased from Vector Biolabs (Malvern, PA, USA).

Techniques: Activity Assay, Transfection, Concentration Assay, Western Blot, Staining, Confocal Microscopy, Incubation, Recombinant, In Vitro

Journal: Cell Death and Differentiation

Article Title: FAF1 mediates regulated necrosis through PARP1 activation upon oxidative stress leading to dopaminergic neurodegeneration

doi: 10.1038/cdd.2016.99

Figure Lengend Snippet: FAF1-mediated PARP1 activation induces energy collapse, mitochondrial depolarization and AIF translocation during oxidative stress. (a and b) Faf1+/+ and Faf1gt/gt MEFs were pretreated with vehicle (DMSO) or DPQ (30 μM) for 1 h and then treated with 500 μM H2O2 for 1 h. Depletion of intracellular energy was determined by measuring the levels of NAD+ (a; n=3) and ATP (b; n=3). (c) Faf1+/+ and Faf1gt/gt MEFs were pretreated with vehicle (DMSO) or DPQ (30 μM) for 1 h and then treated with 500 μM H2O2 for 4 h. The cells were analyzed for mitochondrial membrane depolarization with a Muse analyzer (n=3). (d) Faf1+/+ and Faf1gt/gt MEFs were treated with 500 μM H2O2 for 4 h and subsequently immunostained with the anti-AIF antibody. The nuclei were stained using 4',6-diamidino-2-phenylindole (DAPI) and the cells were analyzed by confocal microscopy. Data (a–c) are expressed as the mean±S.E.M. from three independent experiments. Statistical comparisons were evaluated by ANOVA test followed by Tukey HSD post hoc analysis. ***P<0.001, **P<0.01 and *P<0.05

Article Snippet: In brief, the adeno-associated virus type 1 expressing FAF1 (AAV1-FAF1) was purchased from Vector Biolabs (Malvern, PA, USA).

Techniques: Activation Assay, Translocation Assay, Membrane, Staining, Confocal Microscopy

Journal: Cell Death and Differentiation

Article Title: FAF1 mediates regulated necrosis through PARP1 activation upon oxidative stress leading to dopaminergic neurodegeneration

doi: 10.1038/cdd.2016.99

Figure Lengend Snippet: FAF1 mediates PARP1-dependent necrosis in SH-SY5Y cells during oxidative stress. (a) Left panel: SH-SY5Y cells were transfected with the VC or FAF1 plasmids. Thirty-six hours after transfection, the cells were pretreated with vehicle (DMSO) or DPQ (30 μM) for 1 h and then treated with 500 μM H2O2 for 6 h. Cell death was determined on the basis of LDH release (n=3). Right panel: representative immunoblots show the levels of FAF1 and β-actin expression. (b) Left panel: SH-SY5Y cells were transfected with scRNA or FAF1 siRNA. At 48 h after transfection, the cells were treated with 500 μM H2O2 for 6 h. Cell death was determined on the basis of LDH release (n=3). Right panel: representative immunoblots show the levels of FAF1 and β-actin expression. (c) SH-SY5Y cells were treated with 500 μM H2O2 for the indicated times and were then fractionated into cytoplasmic and nuclear fractions. The fractions were analyzed by immunoblot analysis with anti-FAF1, anti-PARP1 (nuclear marker) and anti-β-actin (cytosolic marker) antibodies. (d) SH-SY5Y cells were treated with 500 μM H2O2 for the indicated times and then the cell lysates were immunoprecipitated with the anti-FAF1 antibody followed by immunoblotting with the indicated antibodies. (e) Left panel: SH-SY5Y cells were transfected with the VC or Flag-FAF1 plasmids. At 36 h after transfection, the cells were treated with 500 μM H2O2 for the indicated times. The cell lysates were immunoblotted with the indicated antibodies. Right panel: the graph shows the result of densitometric analysis of PAR immunoblot (n=3). Quantified data (a, b, e) are expressed as the mean±S.E.M. from three independent experiments. Statistical comparisons were evaluated by ANOVA test followed by Tukey HSD post hoc analysis. ***P<0.001, **P<0.01 and *P<0.05

Article Snippet: In brief, the adeno-associated virus type 1 expressing FAF1 (AAV1-FAF1) was purchased from Vector Biolabs (Malvern, PA, USA).

Techniques: Transfection, Western Blot, Expressing, Marker, Immunoprecipitation

Journal: Cell Death and Differentiation

Article Title: FAF1 mediates regulated necrosis through PARP1 activation upon oxidative stress leading to dopaminergic neurodegeneration

doi: 10.1038/cdd.2016.99

Figure Lengend Snippet: FAF1 promotes dopaminergic neuronal cell death via PARP1 activation in an MPTP mouse model of PD. (a and b) The mice were administered four intraperitoneal injections of MPTP-HCl or saline at 2 h intervals. The mice were killed 24 h after the last injection. (a) Left panel: the subcellular localization of FAF1 was measured by immunofluorescence staining in TH-positive neurons of substantia nigra of mice that were treated with saline or MPTP. The nuclei were stained using DAPI and the tissues were analyzed by confocal microscopy. The white arrows indicate the presence of nuclear FAF1 in TH-positive neurons after MPTP treatment. Right panel: enlarged images of TH-positive neurons were taken from the white box in the merged images. (b) Brain lysates were prepared from the ventral midbrain of saline and MPTP-treated mice and were then subjected to immunoprecipitation with the anti-FAF1 antibody followed by immunoblotting with the indicated antibodies (n=3 per group). (c) The experimental scheme for panel d–f. The mice were injected unilaterally into the substantia nigra (SN) with adeno-associated virus type 1 (AAV1)-FAF1. Two weeks after AAV1-FAF1 injection, the mice were administered MPTP-HCl or saline, at 2 h intervals. The mice were killed 1 day and 7 days after the last MPTP injection and their brain tissues were prepared for western blot (WB) analysis or immunohistochemistry. (d) Left panel: at day 1 after the last MPTP injection, brain lysates were prepared from the ventral midbrain of the AAV1-FAF1-injected (inj.) or non-injected (non-inj.) side of saline- and MPTP-treated mice and were then subjected to immunoblot analysis using the indicated antibodies. Right panel: the graphs show the results of densitometric analysis of PAR and FAF1 immunoblots in left panel (n=3 per group). (e and g) At day 7 after the last MPTP injection, the dopaminergic neurodegeneration was measured by histological analysis for TH-positive (e) and Nissl-positive cells (g) in SN of the AAV1-FAF1 injected (inj.) or non-injected (non-inj.) side of saline- and MPTP-treated mice. Dashed lines represent a region of SN. (f) The graph shows the results of densitometric analysis of TH-stained neurons in e (n=5 per group). (h) The graph shows the counts of Nissl-stained cells in g (n=5 per group). Quantified data (d, f, h) are expressed as the mean±S.E.M. Statistical comparisons were evaluated by ANOVA test followed by Tukey HSD post hoc analysis. ***P<0.001, **P<0.01

Article Snippet: In brief, the adeno-associated virus type 1 expressing FAF1 (AAV1-FAF1) was purchased from Vector Biolabs (Malvern, PA, USA).

Techniques: Activation Assay, Saline, Injection, Immunofluorescence, Staining, Confocal Microscopy, Immunoprecipitation, Western Blot, Virus, Immunohistochemistry