Figure 3.

Knockdown of MEKK−3 and NSY−1 completely suppresses the nuclear localization of SKN−1 and SKN−1−dependent oxidative stress resistance and longevity. (a) The nuclear localization of SKN-1 labeled with GFP in the intestine. SKN-1 transgenic young adult worms were placed on NGM plates that contained either mekk-3, nsy-1, or a combination of mekk-3 and nsy-1 RNAi bacteria for 24 h. The L4440 vector without insertion in bacteria was used as a control. Fluorescent images were captured 4 h after the transfer of worms fed RNAi bacteria on the normal (left) or 20 mM t-BOOH (right) plates. The white arrows indicate SKN-1::GFP. (b) Young adult worms were scored for accumulation of SKN-1::GFP in the intestinal nuclei (n = 30 in each group). (c) Lifespan assay was performed in SKN-1 transgenic worms. Young adult worms were transferred to plates with bacteria containing RNAi plasmids against mekk-3, nsy-1, or both mekk-3 and nsy-1. The L4440 vector without insertion in bacteria was used as a control. The percentage of live animals was plotted at the indicated time points (days). This experiment involved 90 worms in each group. The mean and median lifespan are shown in Table S2. (d) The nuclear localization of SKN-1 S393A mutant labeled with GFP. All procedures were performed in the same manner as described in (a). SKN-1 nuclear localization of each inset was confirmed by using DAPI staining in Figure S4. (e) Quantitative analyses of the fluorescent images of SKN-1 S393A::GFP. All procedures were performed in the same manner as described in (b) (n = 30 in each group). (f) Lifespan assay was performed in SKN-1 S393A transgenic worms. All procedures were performed in the same manner as described in Figure 1b. This experiment involved 90 worms in each group. The mean and median lifespan are shown in Table S2. ** p < 0.01 compared to control. All p values were calculated by chi² tests.