Effect of acclimation to mild hypertonic stress on protein degradation activity.

<p><i>A:</i> Effect of treatment of control and acclimated worms with vehicle only (1% DMSO) or 20 mM chloroquine (CQ) and 100 µM MG-132 on spontaneous aggregation of Q35::YFP. (<i>n</i> = 9–15). *P<0.003 compared to vehicle-treated control worms. **P<0.0001 compared to drug-treated control worms. <i>B:</i> Effect of RNAi silencing of Hos genes on spontaneous Q35::YFP aggregation in control and acclimated worms. Animals were fed bacteria expressing nonspecific (control) dsRNA or dsRNA targeting proteasome (<i>pas-6</i> and <i>rpn-3</i>) and lysosome (<i>vha-13</i>) components, or a putative lysosomal serine carboxypeptidase (F13D12.6). (n = 16–51). *P<0.001 compared to control or acclimated worms fed a nonspecific dsRNA. **P<0.03 compared to unacclimated <i>vha-13(RNAi)</i> worms. <i>C:</i> Percent of red mutant ubiquitin (UbG76V) tagged Dendra2 remaining in body wall muscle cells 24 h after photoconversion in control and 200 mM NaCl acclimated worms exposed to control or hypertonic growth media. Control and acclimated animals were exposed to 400 mM and 600 mM NaCl, respectively. (<i>n</i> = 3–8). *P<0.01 compared to unstressed worms. <i>D:</i> Percent change in ³⁵S-methionine labeled total protein levels in control and acclimated worms treated with 500 µg/ml of cycloheximide for 6 h to inhibit protein synthesis. (<i>n</i> = 3).</p

Effect of elevated glycerol levels on hypertonic stress-induced aggregation of endogenous proteins.

<p><i>A:</i> Effect of increasing NaCl concentrations on motility in control, acclimated, <i>osm-11</i> and acclimated <i>gpdh-1; gpdh-2</i> worms. <i>gpdh-1; gpdh-2</i> mutants lack functional GPDH-1 and GPDH-2 enzymes resulting in greatly reduced glycerol accumulation under hypertonic stress conditions <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034153#pone.0034153-Lamitina2" target="_blank">[12]</a>. (<i>n</i> = 5–18 experiments with 15–60 worms/experiment). <i>B:</i> Left panel, relative insoluble protein in acclimated <i>gpdh-1; gpdh-2</i> worms maintained in either 200 mM NaCl or exposed to 500 mM NaCl for 4 h. Insoluble protein was quantified as a fraction of total protein and is plotted relative to that observed in worms maintained on 200 mM NaCl. (<i>n</i> = 3 experiments with 4000–5000 worms/experiment). Right panel, examples of SDS-PAGE gels of total and detergent insoluble (insol.) proteins isolated from acclimated <i>gpdh-1; gpdh-2</i> worms maintained in 200 mM NaCl or exposed to 500 mM NaCl. <i>C:</i> Left panel, relative insoluble protein in <i>osm-11</i> worms grown under control conditions (51 mM NaCl) or exposed to 700 mM NaCl for 4 h. Insoluble protein was quantified and plotted in the same manner as described in <i>B</i>. (<i>n</i> = 3 samples of 4000–5000 worms/sample). *P<0.03 compared to animals maintained on 51 mM NaCl. Right panel, examples of SDS-PAGE gels of total and detergent insoluble (insol.) proteins isolated from <i>osm-11</i> worms exposed to 51 or 700 mM NaCl.</p

Effect of elevated glycerol levels on aging-induced aggregation of Q35::YFP.

<p><i>A:</i> Whole worm glycerol levels in controls worms, worms acclimated to 200 mM NaCl and <i>osm-11</i> mutant animals. (<i>n</i> = 4 samples of ∼4000 worms/sample). <i>B:</i> Time course of aging-induced Q35::YFP aggregation in control, acclimated and <i>osm-11</i> mutant animals. (<i>n</i> = 7 experiments with 10–15 worms/experiment).</p

Effect of acute hypertonic stress on ³⁵S-methionine incorporation into total protein.

<p>Worms were transferred to 200 mM NaCl agar plates at time 0 and ³⁵S-methionine incorporation into total protein was quantified 20 min and 1, 12 and 48 h after transfer. Values are expressed relative to unstressed control worms (i.e., time 0). (<i>n</i> = 3). *P<0.05 compared to control worms.</p

Effect of elevated glycerol levels on the properties of age-induced Q35::YFP aggregates.

<p><i>A:</i> Fluorescence micrographs of aggregate morphology in body wall muscle cells of control worms, worms acclimated to 200 mM NaCl and <i>osm-11</i> mutant animals. Images were taken from 7-day old adult worms. Scale bar is 10 µm. <i>B:</i> Time course of bleaching and fluorescence recovery in aggregates of young (4-day old) and old (10-day old) adult control, acclimated and <i>osm-11</i> worms (<i>n</i> = 3). <i>C:</i> Aggregate toxicity in control, acclimated and <i>osm-11</i> worms. Toxicity is measured as reductions in motility, which is mediated by body wall muscle cells. (<i>n</i> = 5–12).</p