14-3-3θ immunoprecipitates with Bax in M17 dopaminergic cells.

<p><b>a)</b> Cell lysates from M17 cells were immunoprecipitated with a polyclonal rabbit antibody against Bax or rabbit IgG, and resulting immunoprecipitants were blotted with a monoclonal mouse antibody against 14-3-3θ in top blot. Lysate lane on right is shown at a different exposure time than the immunoprecipitant lanes from the same gel. Blot was reprobed with anti-Bax antibody to verify Bax pulldown (bottom blot). 14-3-3θ shows specific immunoprecipitation with Bax. <b>b)</b> Cell lysates from M17 cells stably transfected with empty vector or 14-3-3θ tagged with the V5 epitope tag were immunoprecipitated with a polyclonal antibody against Bax and then immunoblotted against 14-3-3θ. Both endogenous 14-3-3θ (lower band marked by arrow) and exogenous, tagged 14-3-3θ (higher band marked by arrowhead) were immunoprecipitated with Bax from cells overexpressing 14-3-3θ, and the total amount of 14-3-3θ immunoprecipitated was increased in 14-3-3θ cells compared to empty vector control cells. Lysate lanes on right were run on a separate gel from the immunoprecipitant lanes. Blot was reprobed with anti-Bax antibody to verify pulldown of Bax (bottom blot).</p

Bax inhibition through alternative means is protective against rotenone toxicity.

<p><b>a)</b> M17 cells were pretreated with BIP (0, 200, or 500 µM) for four hours prior to treatment with rotenone at 1 µM. After 48 hours, cell death was assessed by LDH release into the culture media. LDH release into media was normalized to maximal LDH release for each well. Cells treated with BIP were more resistant to rotenone compared to untreated cells. Error bars reflect SEM. Results reflect three independent experiments with at least two replicates per experiment. ***p<0.001 (Bonferroni's multiple comparison test). <b>b)</b> shRNA targeting Bax showed considerable knockdown of Bax protein expression. Naïve M17 cells were infected with a pLKO.1 lentiviral construct containing Bax-specific shRNA sequence or with an empty pLKO.1 lentiviral construct (with no shRNA sequence; C). Infected cells were selected for in the presence of puromycin. Protein lysates from these infected cells were immunoblotted with a polyclonal antibody against Bax (top blot). Immunoblotting against tubulin (bottom blot) shows comparable protein loading. <b>c)</b> pLKO.1 control or Bax-shRNA M17 cells were treated with rotenone at varying concentrations for 48 hours. Cell death was assessed by LDH release. Bax-knockdown cells showed considerable protection against rotenone compared to control cells at all concentrations tested. Error bars reflect SEM. Results reflect three independent experiments with at least two replicates per experiment. ***p<0.001 (Bonferroni's multiple comparison test). <b>d)</b> shRNA targeting Bax also showed knockdown of Bax protein in both empty vector control and 14-3-3θ stable cell lines. Control and 14-3-3θ stable lines were infected with an empty pLKO.1 virus (C) or with the Bax-specific shRNA lentivirus. Protein lysates from these cells were immunoblotted with a polyclonal antibody against Bax and tubulin. <b>e)</b> Empty vector stable and 14-3-3θ stable cells infected with either empty pLKO.1 or Bax shRNA viruses were treated with rotenone at varying concentrations for 48 hours, and cell death was assessed by LDH release. Knockdown of Bax in 14-3-3θ stable cells provided additional reduction of rotenone toxicity. Error bars reflect SEM. Results reflect four independent experiments with at least two replicates per experiment. *p<0.05, **p<0.01, ***p<0.001 (Bonferroni's multiple comparison test).</p

14-3-3θ overexpression reduces rotenone-induced cytochrome C release and caspase 3 cleavage.

<p><b>a)</b> Vector control and 14-3-3θ cells were treated with 5 µM rotenone for 24 hours, and cytosolic fractions were immunoblotted with a mouse monoclonal antibody against cytochrome C. Densitometric quantification includes five independent experiments. Tubulin was used as the loading control for Western blots. Error bars reflect SEM. **p<0.01 (Bonferroni's multiple comparison test). n.s.  =  non-significant. <b>b)</b> Vector control and 14-3-3θ cells were treated with 5 µM rotenone for 24 hours, and cell lysates were immunoblotted with a rabbit polyclonal antibody against cleaved caspase 3. Densitometric quantification includes three independent experiments. Tubulin was used as the loading control for Western blots. Error bars reflect SEM. **p<0.01, ***p<0.001 (Bonferroni's multiple comparison test). n.s.  =  non-significant.</p

14-3-3θ mutant that cannot bind Bax is not protective against rotenone.

<p><b>a)</b> Lysates from stable cells overexpressing either full-length 14-3-3θ or a C-terminally deleted mutant 14-3-3θ (aa1-239) were immunoprecipitated with a polyclonal rabbit antibody against Bax and then immunoblotted with a monoclonal mouse antibody against V5. Blot was reprobed with anti-Bax antibody to verify Bax pulldown (bottom blot). Lysate lanes on right in the Bax blot are shown at a different exposure time than the immunoprecipitant lanes from the same gel. Considerably much less mutant 14-3-3θ was immunoprecipitated with Bax compared to full-length 14-3-3θ. <b>b)</b> Vector control, full-length 14-3-3θ, or mutant 14-3-3θ cells were treated with rotenone for 48 hours. Cell death was assessed by LDH release. While full-length 14-3-3θ cells showed decreased cell death in response to rotenone, cells overexpressing mutant 14-3-3θ showed no protection against rotenone compared to vector control cells. Error bars reflect SEM. Results reflect three independent experiments with at least two replicates per experiment. ***p<0.001 (Bonferroni's multiple comparison test). n.s.  =  non-significant.</p

Rotenone-induced Bax activation is reduced in 14-3-3θ-overexpressing cells.

<p><b>a) Less Bax translocated to mitochondria in 14-3-3θ cells in response to rotenone.</b> After treatment with 5 µM rotenone for 24 hours, vector control and 14-3-3θ cell lysates were subfractionated into cytosolic and mitochondrial fractions and immunoblotted with a polyclonal rabbit antibody against Bax. For each fraction, lanes for vector control and 14-3-3θ cells are from the same gel and exposure time but are separated for clarity with regard to quantification. Bax levels were normalized to tubulin for the cytosolic fraction or cyclophilin D for the mitochondrial fraction. Bax levels for rotenone-treated cells are shown as the relative percentage of the corresponding untreated cells. Densitometric quantification included seven separate experiments. Error bars reflect SEM. *p<0.05, **p<0.01 (one sample t-test). <b>b) Total Bax levels were unchanged with rotenone treatment in either cell line.</b> After treatment with 5 µM rotenone for 24 hours, whole cell lysates were immunoblotted with an anti-Bax antibody. <b>c) Fewer 14-3-3θ cells were positive for activated Bax upon rotenone treatment.</b> After treatment without (i-iv) or with rotenone (v-viii) for 16 hours, vector control and 14-3-3θ cells were fixed in 2% paraformaldehyde and immunostained with a monoclonal mouse antibody against the active Bax conformation (6A7) and a goat Alexa 488-conjugated anti-mouse secondary antibody (i, ii, v, vi). Nuclei were stained with Hoechst 33342 (iii, iv, vii, viii). The number of 6A7-positive cells was quantitated with rater blind to experimental conditions. Error bars reflect SEM. **p<0.01, ***p<0.001 (Bonferroni's multiple comparison test). Scale bar  = 50 µm. <b>d) Rotenone-induced Bax oligomerization was reduced in 14-3-3θ cells.</b> Vector control and 14-3-3θ stable cells were treated with 5 µM rotenone for 24 hours. Mitochondrially-enriched fractions were crosslinked and immunoblotted for oligomers with an anti-Bax antibody. Cyclophilin D served as loading control. Densitometric quantification includes three independent experiments. Error bars reflect SEM. ***p<0.001 (Bonferroni's multiple comparison test). n.s.  =  non-significant.</p

14-3-3θ overexpression reduces rotenone-induced disruption of mitochondrial membrane potential.

<p>Vector control and 14-3-3θ cells were treated with varying doses of rotenone or 10 µM carbonyl cyanide 3-chlorophenylhydrazone (CCCP), a mitochondrial toxin, for 24 hours. Mitochondrial membrane potential was assayed by the JC-1 assay. Ratio of aggregated JC-1 (red) to monomer JC-1 (green) for each condition was normalized to that ratio for the corresponding untreated cells. Results reflect three independent experiments with three replicates per experiment. Error bars reflect SEM. **p<0.01, ***p<0.001 (Bonferroni's multiple comparison test).</p

Dominant negative Akt does not inhibit angiogenin's protective effect against MPP+.

<p>A. Western blot demonstrating reduced Akt phosphorylation in SH-SY5Y cells transfected with the K179M DN-Akt construct. Twenty-four hours following transfection of empty pCMV5 vector (pC) or DN-Akt vector (DN), SH-SY5Y cells were treated with Insulin Growth Factor (50 ng/mL). Thirty seconds after Insulin Growth Factor treatment, cell lysates were collected for Western blotting. Actin was used as a loading control. B. Representative Western blot demonstrating reduced caspase-3 cleavage following angiogenin treatment in SH-SY5Y cells transfected with either empty vector or HA-tagged DN-Akt using Amaxa nucleoporation. Twenty-four hours after transfection, cells were pretreated with angiogenin (100 nM). MPP+ (0.75 mM) was then applied with fresh angiogenin for an additional 24 hours prior to collection. HA-tagged DN-Akt expression was comparable among all DN-Akt-transfected conditions. Actin was used as loading control. C. Densitometric quantification of caspase-3 cleavage bands normalized to actin. Results reflect three independent experiments. *p<0.05, ***p<0.001 (One-way ANOVA with Tukey's post-hoc test). Error bars reflect SEM.</p

Analysis of exogenous fatty acids on the growth of wild-type cells and <i>elo1</i>Δ cells with or without WT α-syn.

<p>SC-galactose liquid media supplemented fatty acids was inoculated with 1×10⁵ cells/ml, incubated for 24 hr at 30°C, and then CFU assay was carried out. The assay was conducted three times.</p><p>Abbreviations, 12:0; lauric acid, 16:0; palmitic acid, 18:0; stearic acid, PHS; phytosphingosine.</p

<i>S. cerevisiae</i> strains and plasmids.

<p><i>S. cerevisiae</i> strains and plasmids.</p

A plasma membrane-binding form of EGFP is toxic, like α-syn, to <i>elo3</i>Δ cells.

<p>(A) Inhibiting ceramide synthetase with FB1, serine palmitoyltransferase with myriocin or blocking C26-VLCFA synthesis in <i>elo3</i>Δ cells drives EGFP-mts1 and EGFP-mts2 into cytoplasmic inclusions. Wild-type cells or <i>elo3</i>Δ cells expressing EGFP-mts1, EGFP-mts2 or EGFP as a control were incubated for 6 h in inducing media containing 5 µM FB1, 1 µM myriocin (or drug vehicle, ethanol) and then imaged by fluorescence microscopy. Approximately 80%, 68%, and 88% of the cells examined exhibited the EGFP-mts1 inclusions as shown in the wild-type strain (+Fum), (+Myr), and in the <i>elo3</i>Δ strain, respectively. 85%, 75%, 70% of the cells examined exhibited the EGFP-mts2 inclusion as shown in the wild-type strain (+Fum), (+Myr), and in the <i>elo3</i>Δ strain, respectively. Plasmids: pAG426GAL-EGFP-mts1, pAG426GAL-EGFP-mts2, and pAG426GAL-EGFP. (B) Growth properties of cells expressing EGFP-mts1, EGFP-mts2, or EGFP were evaluated in a dilution spot assay. EGFP-mts1 or EGFP-mts2 is not toxic to wild-type cells, whereas it was very toxic to FB1-treated (5 µM) wild-type cells and to <i>elo3</i>Δ cells. The control construct EGFP was not toxic to any of the cells. The assay was conducted according to the legend in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015946#pone-0015946-g001" target="_blank">Fig. 1</a>.</p