Knockdown of ceMTM3 causes autophagy in multiples tissues of C. elegans worms at different ages. GFP: LGG-1 worms were cultured on NGM plates containing E. coli cells carrying vector control or ceMTM3 RNAi from the time they were hatched from eggs. GFP-positive puncta (indicated by arrows) represent autophagosomes which are abundant in ceMTM3 RNAi-treated worms. A. Autophagosomes in seam cells of day 2 worm larvae. B. Autophagosomes in muscle cells of day 3 young adult worms. Note that muscle fiber bands are visible in the background. C. Autophagosomes in intestine on day 5 adult worms. D. Average numbers of GFP puncta in 4 seam cells, 60 micrometer-long muscles, and entire intestine. Error bars denote standard deviation, n≥20, p<0.001). E. Western blotting analyses of lipid-bound GFP-LGG-1. Control and ceMTM3 RNAi-treated worms (mixed ages) were extracted in phosphate-buffer saline by sonication. After centrifugation at 800 g for 10 min, the supernatants were collected and subjected to further centrifugation at 100,000g for 1 h. The pellets were extracted in SDS gel sample buffer and directly used for western blotting analysis with an anti-GFP antibody. The position of GFP-LGG-1 is indicated. Equal protein loading is demonstrated by Coomassie blue staining.
Yu et al. BMC Cell Biology 2012 13:28 doi:10.1186/1471-2121-13-28