Expression of 2C and 3C failed to induce complete autophagy in HeLa Cells.

<p>(A) Western blotting of the expression levels of autophagy-related proteins in cells inhibited with 3-MA. HeLa cells were pretreated with 3-MA for 2 h, followed by transfection with viral proteins 2C and 3C. At 24 h after transfection, the cells were subjected to Western blotting using anti-LC3B and Vp1 antibodies. (B, C) Western blotting of the expression levels of autophagy-related proteins in cells inhibited with CQ. HeLa cells were pretreated with CQ for 4 h, followed by transfection with vector or viral proteins 2C and 3C in the absence or presence of CQ. At 24 h after transfection, the cells were subjected to Western blotting using anti-LC3B and HA antibodies. (D) HeLa cells were transfected with either specific shRNA targeting Atg5 or scrambled shRNA. At 12 h after transfection, cells were transfected with vector or viral proteins 2C and 3C. Samples were collected at 24 h after the second transfection and subjected to Western blotting using anti-LC3B and Atg5 antibodies. β-Actin was used as a protein loading control. Representative results are shown with graphs representing the ratio of LC3-II to β-Actin normalized to the control condition. Data are presented as the means from three independent experiments. Significance was analyzed with a two-tailed Student’s <i>t</i> test. **<i>P</i>< 0.01. (E) HeLa cells were cotransfected with the ptfLC3 plasmid and either an empty control vector plasmid or 2C/3C-expression plasmids and incubated for 24 h. Fluorescent signals were visualized by confocal microscopy. The graph shows the quantification of autophagosomes by calculating the average number of dots in 20 cells. Bars, 10μm.