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

Figure 3.

Figure 3

Identification of the effect of apoptin on the changes of autophagy in SMMC-7721 cells. (A,D) To evaluate the changes of average number of LC3 puncta in each cell, SMMC-7721 cells were transfected with pEGFP-LC3 and infected with Ad-VP3 and Ad-mock for 6, 12, 24, and 48 h; Apoptin will cause a significant increase in the amount of LC3 puncta, and the addition of QVD and NAC will cause a significant decrease in the amount of LC3 puncta. (B) Changes in lysosome counting observed by fluorescence microscopy after LTR staining. SMMC-7721 cells were infected with Ad-apoptin or Ad-mock, then stained with LTR at 6, 12, 24, and 48 h post-infection. The number of lysosomes increased from 12 h; the addition of QVD and NAC will cause a significant decrease in the number of lysosomes. (C,E,F) Western blotting analysis of LC3-II and P62 protein expression at different time points on SMMC-7721 cells. The LC3-II and P62 protein level of Ad-apoptin group was higher than Ad-MOCK and control group. (G) After addition of the autophagy inhibitor 3-MA and CQ to Ad-apoptin-infected cells, the growth inhibition rate mediated by apoptin was significantly increased. The scale bar equals 50 μm. Data are means ± SD (*p < 0.05, **p < 0.01, ***p < 0.001) when compared with Ad-mock or controls. Data are shown as the mean ± SD (#p < 0.05) when compared with Ad-apoptin.