ASSNAC effect on lifespan.

<p>ASSNAC effect on lifespan.</p

ASSNAC-induced extension of <i>C</i>. <i>elegans</i> lifespan.

<p>Adult synchronized L4 stage <i>C</i>. <i>elegans</i> (strain CF512) nematodes (20 per plate, 6 plates per treatment) were maintained on NGM agar plates containing ASSNAC (final concentrations: 0.5, 2 and 5 mM) or vehicle (phosphate buffer) and supplemented with OP50 at 20°C. Nematodes were transferred to fresh plates twice a week and their survival was monitored three times a week and presented as fraction of survival (mean ± SE of 6 plates). Experiment was repeated twice and one experiment is presented.</p

ASSNAC protective effect against oxidative stress.

<p><i>C</i>. <i>elegans</i> (N2 strain) in suspension was pretreated with ASSNAC (0.02 to 2.0 mM for 24 hours) and not exposed (control) or exposed to H₂O₂ (3.5 mM for 2 hours). Ninety to 180 nematodes were examined and the percentage of survival is presented as mean ± S.D. of three independent experiments. Percentage of survival in ASSNAC-treated nematodes was found to be significantly different from that in control (p≤0.02) as determined by Kruskal-Wallis Non-Parametric 1-Way ANOVA.</p

ASSNAC effect on glutathione level.

<p><i>C</i>. <i>elegans</i> (strains N2 and CF512) cultured in suspension were treated without (control) or with ASSNAC (2.0 mM for 24 h). Nematodes were collected and both GSH (Reduced) and GSSG (Oxidized) glutathione were determined. Results are presented as mean ± S.D of three independent experiments. The increase in GSH level in ASSNAC-treated nematodes was found to be statistically significant as determined by 2-Way ANOVA with Bonferroni post hoc test (*p≤0.01; **p≤0.001).</p

Effect of ASSNAC on GST enzyme activity.

<p><i>C</i>. <i>elegans</i> strains N2, CF512 and CL2166 were cultured in suspension and treated without (control) or with ASSNAC (2.0 mM for 24 h). Nematodes were collected, lysed and GST activity was determined and presented in Units (μmole/min; mean ± S.D.). GST activity in ASSNAC-treated nematodes was found to be statistically different from control as determined by 2-Way ANOVA with Bonferroni post hoc test (*p≤0.001).</p

Time-dependent effect of ASSNAC on <i>GST-4p</i>::<i>GFP</i> induction.

<p><i>C</i>. <i>elegans</i> CL2166 strain expressing the reporter gene <i>gst-4p</i>::<i>GFP</i> was cultured in suspension in the absence (control) or presence of ASSNAC (10 mM) for the indicated time points and 50–100 nematodes were picked onto slides and analyzed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0194780#pone.0194780.g001" target="_blank">Fig 1</a>. <b>A.</b> Bright-field (upper panel) and fluorescent light (lower panel) images are presented (0, 6 and 18 hours). <b>B.</b> The intensity of the fluorescent signal was quantitated and presented as mean ± S.D. of three independent experiments.</p