Fig. 5

Combination therapy enhances mitochondrial disruption in Huh7-SR cells. A Images of autophagosomes and mitochondria in HCC-SR cells were captured by transmission electron microscopy (TEM). Autophagosomes in the Huh7-SR cells receiving combination therapy contained more damaged mitochondria, suggesting an increase in mitophagy. Scale bar: 5 µm for 6800 ×, 2 µm for 23,000 ×. B Localizations of mitochondria and lysosomes in HCC-SR cells were observed by microscopy. Mitochondria (red) and lysosomes (green) were, respectively, stained with MitoTracker and LysoTracker. Colocalizations of lysosomes and mitochondria were significantly observed in Huh7-SR cells treated with combination therapy, suggesting mitophagy were activated. Scale bar: 5 µm. C The levels of CoxIV, BNIP3, BNIP3L, and β-actin in HCC-SR cells were determined using immunoblotting analysis after cells were treated with sorafenib or combination therapy for 0, 12, 24, 36, 48, and 72 h. The results showed that decreased CoxIV levels (mitochondrial mass) is accompanied by increased BNIP3 and BNIP3L levels over time in the Huh7-SR cells treated with combination therapy. D Immunofluorescence staining of BNIP3/BNIP3L (Red) and CoxIV (Green) 0, 48 and 72 h after Huh7-SR cells received sorafenib or combination therapy. Arrows: co-localization of BNIP3 and Cox IV. Scale bar: 10 µm. E The ATP content was analyzed, which was further decreased in Huh7-SR cells received combination therapy compared to sorafenib alone. F The reactive oxygen species (ROS) levels in HCC-SR cells were measured. Combination therapy caused more accumulation of ROS than sorafenib alone in Huh7-SR cells. G Mitochondrial membrane potential (MMP) levels in HCC-SR cells were determined using JC-1 staining. JC-1 aggregates (red) represent the high membrane potential while the JC-1 monomers (green) represent the low membrane potential. The result suggested that combination therapy caused a further decrease in MMP levels in Huh7-SR cells compared to sorafenib alone. Scale bar: 50 µm. Cells in A, B, and E–G were treated as indicated for 48 h. ****p < 0.0001. ns represents not statistically significant. Full-length blots are presented in Additional file 6