Fig. 3

Glu + NA + NAC combinatorial treatment effects on C. elegans gas-1(fc21) worms’ mitochondrial physiology. (A) In vivo fluorescence analyses of mitochondrial content, mitochondrial oxidant burden and mitochondrial membrane potential were performed by terminal pharyngeal bulb (PB) relative fluorescence microscopy quantitation of MitoTracker Green FM (MTG), MitoSOX (SOX) and TMRE, respectively. gas-1(fc21) mutant synchronized young adult worms were treated for 24 h with Glu + NA + NAC combination therapy, or its individual and pairwise components, were compared with buffer-only treated gas-1(fc21) and N2 (WT) worms, and normalized to buffer-only gas-1(fc21) worms. Significant differences in mean fluorescence intensity between strains under different experimental conditions were assessed by mixed-effect ANOVA to account for potential batch effect due to samples being experimentally prepared, processed and analyzed on different days by including a batch random effect in the model. Statistical significance threshold was set at P < 0.05, and statistical analyses were performed in SAS 9.3. P-value conveys the significance of the difference between untreated N2 and untreated gas-1(fc21) (strain effect) or the difference between gas-1(fc21) plus drug(s) and untreated gas-1(fc21) (treatment effect). For each parameter, each drug treatment assay was repeated in 3 to 10 independent trials, with n = 50 worms per trial. Bars and error bars convey mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001 versus concurrent gas-1(fc21) buffer control. (B) Feeding RNA interference (RNAi) knockdown of gas-1 (K09A9.5) in an hsp-6p::gfp labeled wild-type reporter worm strain resulted in significantly elevated UPR^mt in first day young adult stage worms relative to empty vector (L4440) RNAi control, which was significantly reduced by treatment from early development (L1 stage) with glucose (Glu) or N-acetylcysteine (NAC) and any pairwise or triple combinatorial regimens in which they were present. Nicotinic acid (NA) alone did not reduce UPR^mt in this model. hsp-6p::gfp C. elegans was used as a positive control. All tests were carried out using three biological replicate independent trials, with approximately 300 worms per condition in each replicate. Boxes depict 10th and 90th percentile for the normalized fluorescence intensity. Whiskers depict minimum and maximum values for each condition. Statistical analysis was performed by unpaired t-test (**, P < 0.01; ***, P < 0.001) for each comparison, as indicated.