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. 2020 Jun 25;10:1026. doi: 10.3389/fonc.2020.01026

Figure 6.

Figure 6

Effects of ROS toward apoptosis and autophagy in apoptin-induced SMMC-7721 cells. (A) ROS levels in SMMC-7721 cells were analyzed by flow cytometry after DHR staining. Upon treatment with QVD, ROS levels in the QVD+apoptin group decreased to varying degrees. Upon addition of autophagy inhibitor 3-MA and CQ, ROS levels in the CQ+Ad-apoptin group was significantly higher than that in the untreated apoptin group starting from 24 h post-infection; and ROS levels in the 3-MA+Ad-apoptin group was significantly higher than that in the untreated apoptin group starting at 48 h post-infection. (B) After addition of the ROS inhibitor NAC to Ad-apoptin-infected cells, the growth inhibition rate mediated by apoptin was significantly decreased. (C,D) SMMC-7721 cells apoptosis were analyzed by flow cytometry after Annexin-V FITC/PI staining. The apoptosis level of SMMC-7721 cells infected with Ad-apoptin was significantly higher than that of other groups; the addition of NAC will cause a significant decrease in the apoptosis level of SMMC-7721 cells infected with Ad-apoptin. (E,F) Western blotting analysis of cleaved-PARP and cleaved-Caspase-3 in SMMC-7721 cell extracts. Inhibition of ROS can decrease apoptin-induced apoptosis in liver cancer cells and then increases over time. (G,H) Western blotting analysis of LC3-II and P62 in SMMC-7721 cell extracts. Inhibition of ROS in apoptin-treated liver cancer cells can significantly reduce autophagy levels. Data are shown as mean ± SD (*p < 0.05, **p < 0.01, ***p < 0.001) when compared with Ad-mock or controls. Data are shown as mean ± SD (#p < 0.05, ##p < 0.01) when compared with Ad-apoptin.