Hepatitis B Virus Disrupts Mitochondrial Dynamics: Induces Fission and Mitophagy to Attenuate Apoptosis
Figure 2
HBV induces mitochondrial translocation of Parkin and stimulates mitophagy-related genes.
(A) Huh7 cells transfected with HBV construct were prestained with MitoTracker (Mito, red) and subsequently, immunostained with anti-Parkin (green) and anti-HBsAg (white) antibodies. Nuclei are demarcated with white dots. Transfected (+) and untransfected (−) cells are marked. In the zoomed images, yellow color indicates the accumulation of endogenous Parkin recruited to the mitochondria. Quantification of fluorescence intensity of Parkin aggregates on the mitochondria in HBV-expressing cells. (B) The cytosolic (Cyto) and mitochondrial (Mito) fractions were isolated from HepAD38 cells grown in the absence or presence of tetracycline for 48 h. Cellular fractions were analyzed by Western blotting with antibodies specific for the indicated proteins. Fractions: WCL, whole cell lysates; Cyto, purified cytoplasm; Mito, purified mitochondria. Organelle markers: VDAC1, mitochondria; GAPDH, cytoplasm. (C) Parkin protein in whole cell lysates of HBV-expressing cells was immunoprecipitated by anti-Parkin antibody, followed by immunoblotting (IB) with anti-ubiquitin (Ub) antibody. Normal rabbit IgG was used as a control for immunoprecipitation (IP). Western blot analysis for β-actin indicates equivalent amount of cell lysates for IP (C and D). (D) Mfn2 protein in whole cell lysates extracted from HepAD38 cells transfected with non-targeting (NT) and Parkin (P) siRNA, respectively, for 48 h was immunoprecipitated by anti-Mfn2 antibody, followed by immunoblotting (IB) with anti-ubiquitin (Ub) antibody. Normal mouse IgG was used as a control for immunoprecipitation (IP). The protein expression was analyzed by Western blotting with antibodies specific to Mfn2, Parkin, and β-actin proteins. (E and F) HepAD38 and HepG2 cells were grown in the absence or presence of tetracycline (1 µg/ml) for 12 h. (E) Intracellular mRNA levels of Parkin, PINK1, and LC3B were analyzed by real-time qRT-PCR. GAPDH was used to normalize changes in Parkin, PINK1, and LC3B gene expression. (F) The protein expression was analyzed by Western blotting with antibodies specific for the indicated proteins. β-actin was used as a loading control (F and G). The relative intensity of ATF4, Parkin, PINK1, and LC3B protein expression normalized to β-actin was analyzed by ImageJ software. (G) Whole cell lysates extracted from Huh7 cells with empty vector (Mock) and those with HBV construct for 48 h were analyzed by Western blotting with antibodies specific to the indicated proteins.