Figure 2. Autophagy increases transiently at 1 week after rapamycin treatment initiation. Rapamycin increased levels of autophagic markers LC3 II/I (A) and ATG5 (B) in old hearts. * p <0.05 vs. old Ctrl; # p <0.05 vs. 1wk; + p<0.05 vs. 2 wk. (C) Immunoblotting showed increased LC3 II/LC3 I ratio and Atg5 levels after 1-week rapamycin treatment which returned towards the control level after 2 weeks. (D-G) Rapamycin induced accumulation of LC3 II after leupeptin injection at 1 week (D, G) but not 2 weeks (E, G) or 10 weeks (F, G) after treatment initiation. * p <0.05 vs. 1wk Saline; # p<0.05 vs. Ctrl Leupeptin. Immunoblotting of ULK1 (H) showed that rapamycin inhibited ULK phosphorylation at S757 at 1-week (I) but not 2-week (J) and 10-week time points (K). n≥5/group. * p <0.05 vs. Ctrl. Sal, saline; Leu, leupeptin. Data are represented as mean ± SEM.