(A) Neonatal mouse ventricular cardiomyocytes (NMVCs) were cultured under hypoxic conditions (5% CO₂ and 95% nitrogen at 3 l/min) in the absence of glucose for 14 hours at 37°C followed by reoxygenated for 4 hours with 5% CO₂ and 95% humidified air in medium containing glucose (hypoxia/reoxygenation [H/R]) or under normoxic conditions. Myocytes were then harvested and cellular lysates were immunoblotted for BAG3, cleaved caspase-3, B cell lymphoma 2 (Bcl-2), and lysosomal-associated protein 2 (LAMP-2). β-Actin served as protein loading control. Three independent cultures of NMVCs were exposed to either H/R or control conditions in each experiment. Each experiment was repeated 3 times in order to have n = 9 for each intervention. (A) Representative immunoblot of one experiment (n = 3 for I/R and control) in which NMVCs were subjected to normoxia (control) or H/R. (B–E) Quantification of Western blots for levels of BAG3, Bcl-2, cleaved caspase-3, and LAMP-2 (n = 9 for both H/R and control). Data were normalized to the protein levels measured under normoxic conditions and analyzed using a Student’s t test. (F) BAG3 levels were knocked down using a siRNA by infecting NMVCs with either an adenovirus driving BAG3 (Ad-siBAG3) or a GFP control (Ad-GFP). Cells were cultured for 3 days after which they were harvested and immunoblots were performed. Three independent cultures of NMVCs were exposed to either Ad-siBAG3 or Ad-GFP (control) in each experiment. Each experiment was repeated 3 times in order to have n = 9 for each intervention. A representative Western blot demonstrating that BAG3 knockdown resulted in a significant decrease in BAG3 levels associated with a decrease in Bcl-2 and LAMP-2 and an increase in cleaved caspase-3 is shown. (G–J) Quantification of Western blots for BAG3, Bcl-2, cleaved caspase-3, and LAMP-2 (n = 9). Data were normalized to the protein levels measured in NMVCs infected with control Ad-GFP and were analyzed using a Student’s 2-tailed t test. Data are presented as mean ± SEM