Figure 2From: Nonstructural proteins 2C and 3D are involved in autophagy as induced by the encephalomyocarditis virusNonstructural proteins 2C and 3D increased autophagic activity and the formation of autophagosomes. (A) Western blotting analyses of LC3, p62 and β-actin in BHK-21 cells that were transfected individually with an HA-tagged EMCV protein-expressing plasmid. HA, the BHK-21 cells transfected with pCMV-HA; Mock, the untransfected BHK-21 cells. The band intensities of LC3-II, p62 and β-actin were quantified, and the relative ratios of LC3-II/β-actin and p62/β-actin are shown in the lower blots. (B) The BHK-21 cells were transfected with pCMV-HA-2C, pCMV-HA-3D, pCMV-HA-VP1 or pCMV-HA and mock (untransfected BHK-21 cells) for 48 h, and they were fixed, processed, and imaged by transmission electron microscopy (TEM). The morphologically characteristic double-membrane vesicles are indicated by black arrows in the relevant areas. Magnification, 10,000×; scale bars, 2 μm. (C) A quantification of the number of autophagosome-like vesicles per cell profile in BHK-21 cells that were transfected with 2C-, 3D-expressing plasmid, pCMV-HA-VP1 or pCMV-HA and mock (untransfected BHK-21 cells) Data are the means ± SD (error bars) for 8 cells per experimental condition from three independent experiments. ***p < 0.001, compared with the control cells. (D) BHK-21 cells were transfected with 10, 20, 30 or 40 pmol Beclin1-siRNA or 40 pmol control-siRNA, and then Beclin1 protein was detected by western blotting at 48 h post-transfection. Mock, the untransfected BHK-21 cells. (E) BHK-21 cells transfected with 40 pmol Beclin1-siRNA or control-siRNA. After 48 h, the knockdown cells were reseeded, transfected with pCMV-HA-2C, pCMV-HA-3D or pCMV-HA for an additional 48 h and processed for Western blotting analysis. The band intensities of LC3-II, p62 and β-actin were quantified, and the relative ratios of LC3-II/β-actin and p62/β-actin are shown in the lower blots.Back to article page