Figure 7

Rapamycin treatment restored the lysosome content of cardiomyocytes. (A) NRVCs were transduced with Ad-GFP-LC3 for 48 hours, incubated with LysoTracker (red), and observed by confocal microscopy. Representative image shows the localization of lysosome (red) and autophagy puncta (green) in Nef expressing cells. (B) Quantification of lysosomes and autophagosomes in cardiomyocytes (n = 4 wells/group, *P < 0.05, vs Ad-null; ^† P < 0.05, vs Ad-null; ^# P < 0.05, vs Ad-null; ^$ P < 0.05, vs Ad-null; ^¶ P < 0.05, vs Ad-null and Ad-Nef; ^‡ P < 0.05, vs Ad-Nef). (C) Representative images showed that rapamycin treatment reduced cytosolic accumulation of TFEB protein in Nef transduced cardiomyocytes. NRVCs were transduced with Ad-null or Ad-Nef for 48 hours and treated with rapamycin for 12 hours. Cells were fixed with 4% PFA and immunocytochemistry was done with TFEB (green) and Nef (red) antibodies respectively.