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

ASSNAC effect on lifespan.

<p>ASSNAC effect on lifespan.</p

The effect of anti-coagulant and anti-platelet agents on testicular arterial blood flow following DXR treatment.

<p>Blood flow was measured by the PW Doppler mode and quantified by analyzing the VTI using the appropriate VisualSonics software. (<b>a</b>) Blood flow of testicular arteries was continuously monitored by PW Doppler in mice pre-treated with LMWH (clexane; 100µg/mouse), before and following DXR (8mg/kg BW; n=8, n=number of imaged arteries) injection and analyzed 3, 6, 9, 12 and 15 minutes afterwards. Graphic illustration of testicular arterial blood flow; points represent percent of control (dashed line; mean±SEM), (*)-significantly different from DXR treatment values (P<0.05). (<b>b</b>) Blood flow of testicular arteries was continuously monitored by PW Doppler in mice pre-treated with eptifibatide (integrilin; 75µg/mouse), before and following DXR (8mg/kg BW; n=9, n=number of imaged arteries) injection and analyzed 3, 6, 9, 12 and 15 minutes afterwards. Graphic illustration of testicular arterial blood flow; points represent percent of control (dashed line; mean±SEM), (*)-significantly different from DXR treatment values (P<0.05).</p

Effect of Doxorubicin on platelet aggregation and adhesion.

<p>(A) PRP was pre-incubated for 15 minutes with increasing concentrations of DXR then aggregation was induced by ADP (5 µM) and maximal aggregation is presented as mean ± SD; statistical analysis was tested by ANOVA (p<0.001; n=9). (B) Whole blood was pre-incubated for 15 minutes with increasing concentrations of DXR then subjected to the Impact-R test and both surface coverage and average size of the aggregates are presented as mean ± SD (n=5).</p

DXR induces testicular vascular changes.

<p>Histological sections of testes from saline or DXR (5 mg/kg) treated mice were immunohistochemically stained with Hoecst 33342 (nuclei labeling; blue) and anti CD34 primary antibody (1:200) followed by alexa555 anti-rat secondary antibody (red; 1:400). Saline (A,C) and DXR (B,D) at one week (A,B) or one month (C,D) after treatment. Bar=240µm.</p

Platelet adhesion to DXR-treated aortic endothelial cells.

<p>Confluent endothelial cells were exposed to growth medium without (A) or with Doxorubicin (B; 100 µM) for 4 hr followed by exposure to whole blood for 5 minutes at 37°C under defined shear rates (750 s^-1) and then fixed and platelets were stained by immunohistochemistry kit using monoclonal antibody against the platelet integrin CD41a.</p