Research Paper Volume 13, Issue 10 pp 13474—13495

Figure 2. G. lucidum extends nematode lifespan by inducing autophagy. (A–D) Effects of G. lucidum (GL) in wild-type and mutant C. elegans. Synchronized L4 larvae of (A) wild-type (WT) N2 C. elegans or mutant strains lacking (B) atg-18, (C) daf-16, or (D) sir-2.1 were maintained on agar plates spread with GL (2 mg/plate) or rapamycin (Rapa, 400 μM), and survival was monitored based on motility (see also Supplementary Table 1). (E) GL induces autophagy in C. elegans. Transgenic DA2123 C. elegans expressing GFP::LGG-1 were treated as above for 3 days, prior to observation under fluorescence microscopy. Approximately 50 cells were examined per treatment. (F) Quantification of fluorescent GFP::LGG-1 puncta following GL treatment based on the experiments shown in (E). (G) GL treatment increases GFP::LGG-1 levels in DA2123 worms as revealed by Western blotting. Membranes were incubated with both anti-GFP and anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibodies, prior to signal detection. (H) Quantification of Western blot signals shown in (G) after normalization against GAPDH. *p<0.05; **p<0.01; ***p<0.001.