Mitochondrial OXPHOS dysfunction and excess ROS accumulation in HBV-replication hepatoma cell lines (IMAGE)
Caption
(A) OCR was measured by Seahorse analyzer to determine basal respiration, ATP-linked respiration, proton leak, and maximal respiration capacity in HepG2 and HBV-replication hepatoma cell lines (HepG2.2.15 and HepAD38) with or without antiviral agent (ETV) treatment. (B) MMP was detected using JC-1 dye by flow cytometry in HepG2, HepG2.2.15, and HepAD38 with or without ETV treatment. A decrease in the JC-1 aggregates/monomers ratio indicated declined MMP, mitochondrial depolarization, or mitochondrial dysfunction. (C–E) Intracellular ATP levels and mitochondrial complex Ⅰ or Ⅲ activity were assayed in HepG2, HepG2.2.15, and HepAD38 with or without ETV treatment. (F) Cellular ROS levels were determined using DCFH-DA dye by flow cytometry in HepG2, HepG2.2.15, and HepAD38 with or without ETV treatment. Higher MFI of DCFH-DA dye indicated more ROS production in cells. (G) Cellular ROS levels were detected using DCFH-DA dye by flow cytometry in HepG2, HepG2.2.15, and HepAD38 cells with or without treatment of mitochondria-targeted ROS scavenger Mito-TEMPO after 24, 48, and 72 h of incubation period. All data were shown as mean ± standard error of the mean from at least 3 independent experiments. P-values < 0.05 were considered statistically significant (∗P < 0.05, ∗∗P < 0.01, or ∗∗∗P < 0.001 between two indicated groups). OCR, oxygen consumption rate; ETV, entecavir; MMP, mitochondrial membrane potential; ATP, adenosine triphosphate; ROS, reactive oxygen species; MFI, mean fluorescence intensity; HBV, hepatitis B virus.
Credit
Rui Song, Shasha Yu, Xueyan Chen, Ning Ling, Dachuan Cai, Hong Ren, Min Chen
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