Figure 4. MES-induced phenotypes are not observed in AMPK-kinase LKB1 homolog par-4 mutant worms.

(A–D) par-4 mutant worms (A, B: it47; C, D: it57) were treated once a day with MES (0.1 ms, 2 V/cm, 55 pps) for 20 min at larval stage. After the last treatment, worms were incubated under (A, C) heat stress (30°C) or (B, D) oxidative stress (4 mM paraquat) condition. Worms survival were checked every 24 hr. Each time point is the average of three independent experiments using 80–100 animals per group. (E–H) par-4 mutant worms (E, F: it47; G, H: it57) were bred on NGM plates and treated once a day with MES for 20 min at larval stage. After the last treatment, total RNA was extracted and subjected to quantitative real-time PCR. The mRNA levels were normalized to the level of β-actin (internal control). Data are presented as mean ± SE. n.s. versus control, assessed by unpaired t-test. (I) par-4(it47, it57) worms were bred on NGM plates containing D-glucose (10 mM) and treated once a day with MES for 20 min at larval stage. After the last treatment, worms were stained with Nile red and observed using a fluorescent microscope. (J, K) Relative fluorescence intensity of Nile red for (J) it47 and (K) it57 was quantified. Data are representative of two independent experiments using 40–50 animals per group. Data are presented as mean ± SE. ^***P<0.001 versus control without D-glucose, n.s. versus control with D-glucose, assessed by one-way ANOVA. (L, M) par-4 mutant worms (L: it47; M: it57) were bred on NGM plates containing D-glucose (10 mM) and treated once a day with MES for 20 min at larval stage. After the last treatment, total RNA was extracted and subjected to quantitative real-time PCR. The mRNA levels were normalized to the level of β-actin (internal control). Data are presented as mean ± SE. n.s. versus control, assessed by unpaired t-test. n.s., not significant. For mRNA analysis (E–H and L–M), data shown are representative of two independent experiments using approximately 200 animals per group.