Figure 4. Effects of acacetin on mitophagy kinase proteins and mitochondrial membrane potential in H9C2 cardiac cells. (A) Western blot of mitochondrial LC3II, LC3I and relative LC3II/LC3I ratio in mitochondrial proteins of cells treated without (control) or with D-galactose (D-gal) exposure or D-galactose plus 0.3, 1 or 3 μM acacetin for 72 h. (B) Western blot and relative level of mitochondrial PINK1 in cells treated as in (A). (C) Western blot and relative level of mitochondrial Parkin in cells treated as in (A). (D) Representative graphs of flow cytometry for determining mitochondrial membrane potential in cells stained with JC-1 (2 μM) in cells treated without (control) or with D-galactose (20 mg/mL) or D-galactose plus 3 μM acacetin. (E) Percentage of mitochondrial membrane potential depolarization (δψM) in cells treated as A. (n = 5, **P< 0.01 vs. control. #P < 0.05, ##P < 0.01 vs. D-gal).