To determine whether BAG3 translocated to the nucleus during hypoxia/reoxygenation (H/R), neonatal mouse ventricular cardiomyocytes (NMVCs) were exposed to H/R (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) or normoxic conditions and infected with an adenovirus encapsulated with a siRNA (Ad-siBAG3) to decreased BAG3 levels or with an adenovirus encapsulated with GFP (Ad-GFP control). Three independent cultures of NMVCs were used in each experiment in order to have n = 3 for each intervention. NMVCs were fixed and stained with a BAG3 antibody, an α-actinin antibody, or the nuclear counter-stain DAPI. (A) Representative confocal images of BAG3 localization in NMVCs that were positive for DAPI and α-actinin. Scale bars: 10 μM. Both H/R and BAG3 knockdown with Ad-siBAG3 appear to increase the presence of BAG3 in the nucleus when compared with NMVCs treated under control conditions. (B) To confirm the visual observation that BAG3 translocated to the nucleus during the stress of H/R or after knockdown with siBAG3, cytoplasm and nuclear fractions of NMVCs were prepared using a NE-PER nuclear and cytoplasmic extraction kit (ThermoFisher Scientific). Western blot of BAG3 in cytoplasmic and nuclear fractions obtained from Ad-GFP and BAG3 knockdown (Ad-siBAG3) NMVCs subjected to normoxic (control) or H/R incubations demonstrates that β-tubulin was present in all 3 cytosol fractions but not in the nuclear fraction, whereas histone was present in all 3 nuclear fractions but not in the cytosol, suggesting adequate separation of the two fractions. (C) Quantification of Western blots of nuclear extracts demonstrates that BAG3 levels (normalized to histone) in nuclear extracts increased following BAG3 knockdown (Ad-siBAG3) or after H/R. Two-way ANOVA with Bonferroni multiple comparison adjustments were used to assess differences across the 4 investigational groups.