Figure 1.
Effects of the ethanol extract of red mold dioscorea (RMDE) on oxidative-stress resistance of wild-type (WT) Caenorhabditis elegans N2.
(A) Dose response for juglone exposure. Synchronized WT L1 larvae were pretreated with the RMDE (1, 10, and 50 µg/mL) or 0.1% DMSO as the solvent control for 72 h at 20°C. Subsequently, adult worms were subjected to oxidative-stress assays. For the oxidative-stress assays, RMDE-treated (n = 100) and control (0.1% DMSO, n = 100) adult worms were exposed to 0, 10, 50, 100, 150, 150, 250, and 500 µM juglone for 3.5 h at 20°C and then scored for viability. (B) Survival of RMDE-treated and control adult worms exposed to 250 µM juglone for 3.5 h at 20°C. The test was performed three times. Error bars represent the standard error, and differences compared to the control (0 µg/mL, 0.1% DMSO) were considered significant at p<0.05 (*), p<0.01 (**) by one-way ANOVA and the LSD post-hoc test.
Figure 2.
Effects of the ethanol extract of red mold dioscorea (RMDE) on reactive oxygen species (ROS) accumulation in Caenorhabditis elegans.
(A) Synchronized WT L1 larvae were pretreated with the RMDE or 0.1% DMSO as the solvent control for 72 h at 20°C. Subsequently, intracellular ROS were analyzed and the results are presented as relative fluorescence units (RFU) of 20 individual worms (n = 160). (B) Oxidative-stress resistance of the mev-1 mutant. Synchronized mev-1 L1 larvae were pretreated with the RMDE or 0.1% DMSO as the solvent control for 72 h at 20°C. Subsequently, mutant worms were subjected to oxidative-stress assays. RMDE-treated (n = 76) and control (0.1% DMSO, n = 101) worms were exposed to 250 µM juglone for 3.5 h at 20°C and then scored for viability. The test was performed three times. Error bars represent the standard error, and differences compared to the control (0 µg/mL, 0.1% DMSO) were considered significant at p<0.05 (*), p<0.01 (**), and p<0.001 (***) by one-way ANOVA and the LSD post-hoc test.
Figure 3.
Effects of the ethanol extract of red mold dioscorea (RMDE) on DAF-16.
(A) Survival of daf-16 deletion mutants under oxidative stress. Synchronized L1 larvae were pretreated with the RMDE (10 µg/mL, WT N2. n = 100; daf-16(mgDf50), n = 98; daf-16(mu86), n = 120) or 0.1% DMSO as the solvent control (WT N2, n = 100; daf-16(mgDf50), n = 108; daf-16(mu86), n = 143) for 72 h at 20°C. Subsequently, worms were subjected to oxidative-stress assays. Worms were exposed to 250 µM juglone for 3.5 h at 20°C and then scored for viability. (B) Effect of RMDE on subcellular DAF-16 localization. Immediately after hatching, age-synchronized transgenic C. elegans L1 larvae (TJ356) were pretreated with the RMDE (10 µg/mL) and 0.1% DMSO as the solvent control for 72 h at 20°C. The subcellular distribution of DAF-16 was examined in approximately 20 animals per condition by fluorescence microscopy. Worms were classified into the categories of “cytosolic” (left panel), “intermediate” (middle panel), and “nuclear” (right panel) according to their localization phenotypes. Comparative evaluation of the subcellular distribution of DAF-16 in the groups (C) without and (D) with oxidative stress. For the oxidative-stress assays, RMDE-treated (10 µg/mL) and control (0.1% DMSO) adult worms were challenged with 50 µM juglone for 5 min, and then their fluorescence intensity was scored. The results are presented as the percentages of each localization phenotype in the total worms tested. The test was performed three times. Error bars represent the standard error, and differences compared to the control (0.1% DMSO) were considered significant at p<0.01 (**) by one-way ANOVA and the LSD post-hoc test. ns, no significant.
Figure 4.
Effects of the ethanol extract of red mold dioscorea (RMDE) on the expression of superoxide dismutase (SOD).
(A). Effects of RMDE on the expression of SOD-3::green fluorescent protein (GFP). Immediately after hatching, age-synchronized, transgenic L1 worms of the CF1553 strain (sod-3:: GFP) were treated with 10 µg/mL RMDE or 0.1% DMSO as the solvent control for 72 h at 20°C, followed by 150 µM juglone exposure for 1 h. Subsequently, the total GFP fluorescence of each whole worm was quantified by Image-Pro Plus software. Data shown are the average number of pixels in transgenic Caenorhabditis elegans (n = 20) at each indicated treatment. (B) Effects of RMDE on the SOD-3 mRNA level. Synchronized WT N2, daf-16(mgDf50) mutant, and CF1553 strain L1 larvae were pretreated with 10 µg/mL RMDE or 0.1% DMSO as the solvent control for 72 h at 20°C and then total RNA was extracted. mRNA level of SOD-3 was determined by quantitative real-time RT-PCR. SOD-3 mRNA levels were normalized to the expression of AMA-1. The fold change was normalized to that observed in untreated control C. elegans samples. The test was performed three times. Error bars represent the standard error, and differences compared to the control (0.1% DMSO) were considered significant at p<0.05 (*), p<0.01 (**) by one-way ANOVA and the LSD post-hoc test. ns, no significant.