Figure 4. Sulforaphane increases stress resistance. (A) Approximately 100 N2 wild-type C. elegans L4 larvae per group were transferred to liquid NGM medium containing 120 μM juglone (Jug), either alone together with 100 μM sulforaphane (SF), whereas the negative control (Neg. CO) did not contain either. The worms were maintained at 20° C for 24 h, followed by counting the number of surviving worms, which was set as 100 in the negative control, and the percentage of surviving control and sulforaphane worms was calculated related to the negative control (% survival). (B) Approximately 100 wild-type C. elegans L4 larvae were transferred to NGM agar medium containing 100 μM sulforaphane (SF) or not (CO), followed by incubation at 35° C for 4 h, 8 h and 12 h, as indicated. Thereafter, the number of surviving worms was evaluated and is presented as the % survival. (C) Approximately 100 L4 C. elegans N2 wild-type larvae were grown in 100 μM sulforaphane (SF) or were left untreated (CO). Ten days later, 20 adult worms were selected, washed and incubated in 5 μM DHE in M9 buffer in the dark at 20° C for 1 h. After washing, the worms were mounted on a glass slide, paralyzed with a drop of 10 mM sodium azide, and the total fluorescence of each worm was analyzed by fluorescence microscopy, where that the intensity of fluorescence was dependent on intracellular oxidation of DHE by superoxide/ROS to form 2-hydroxyethidium (2-EOH). Representative images at 100× magnification are shown, and the scale bar indicates 0.1 mm. The fluorescence intensity was analyzed with ImageJ, and the fluorescence of the control was set as 1. (D) Fifty adult C. elegans N2 wild-type worms were treated and selected as described above. After washing, proteins were harvested, and 50 μl of protein lysate per group was incubated with a 20 μM concentration of the fluorescent dye CM-H2DCFDA for 30 min at 20° C in the dark. CM-H2CFDA is oxidized by superoxide/ROS into the fluorescent dye DCF, which was quantified through the use of a microplate reader with excitation at 485 nm and emission at 520 nm. The data are expressed as the mean ± SD. *P <0.05, **P<0.01.