Fig. 1.

CryAB^R120G expression is a potent activator of cardiomyocyte autophagy. (A) Representative low (×5,000; Upper) and high (×20,000; Lower) magnification images of NRVMs 5 days after expression of CryAB^WT or CryAB^R120G. Aggresomes are evident in CryAB^R120G-expressing cells (asterisks) as are extensive perinuclear autophagosomes (arrows). (B) Despite extensive induction of autophagy, there is no appreciable change in cell viability 5 days postinfection. (C) NRVMs were transiently transfected with a GFP-LC3 construct and then infected with WT or mutant CryAB. Twenty-four hours later, autophagy was quantified as the number of punctate-positive cells divided by the total number of GFP+ cells. (D) Representative images of NRVMs (two examples of each) infected with CryAB and processed for mTOR immunocytochemistry. mTOR is distributed throughout the cytoplasm in NRVMs expressing WT CryAB. In contrast, CryAB^R120G triggered formation of perinuclear aggregates that stain for both mTOR and crystallin.