Fig. 6

NS38 degrades N proteins in an autophagy-dependent manner. A Inhibitors of autophagy progress block NS38-mediated degradation of N protein. EPC cells were seeded in 6-well plates overnight and co-transfected the indicated plasmids (1 μg each). At 18 h post-transfection, the cells were treated with DMSO, MG132 (20 μmol/L), 3-MA (2 mmol/L), Baf-A1 (100 nmol/L), or CQ (100 μmol/L) for 6 h. The cell lysates were subjected to immunoblotting (IB) with the indicated antibodies (Abs). B–D The NS38-induced N protein degradation is rescued by 3-MA, Baf-A1, and CQ on a dose-dependent manner. EPC cells were seeded in 6-well plates overnight and co-transfected with the indicated plasmids. At 18 h post-transfection, the cells were treated with 3-MA (1 or 2 mmol/L), Baf-A1 (50 or 100 nmol/L) or CQ (50 or 100 μmol/L) for 6 h. Then, the cells were harvested for IB with the indicated Abs. E, F The presence of NS38 or N-only does not affect autophagy flux. EPC cells were seeded in 6-well plates and transfected with N-Myc or NS38-HA (0.5, 1, or 1.5 μg). At 18 h post-transfection, the cells were treated with DMSO or Baf-A1 (100 nmol/L) for 6 h. Then, the cells were harvested for IB with the indicated Abs. G, H Co-expression of NS38 and N protein specifically activates autophagy. EPC cells were seeded in 6-well plates overnight and co-transfected the indicated plasmids (1 μg each). At 18 h post-transfection, the cells were treated with DMSO, 3-MA (2 mmol/L), Baf-A1 (100 nmol/L), or CQ (100 μmol/L) for 6 h. The cell lysates were subjected to IB with the anti-LC3, anti-Myc, anti-HA and anti-β-actin Abs, respectively. EBSS was used to exclude the effect of non-specific cellular autophagy activation on N protein expression. I–L Co-expression of NS38 and N protein induces aggregation of LC3. EPC cells were plated onto coverslips in 6-well plates and co-transfected with 1 μg pCS2-mCherry, NS38-mCherry, or N-mCherry plus 1 μg LC3-GFP. After 24 h, the cells were fixed and observed by confocal microscopy. Red signals represent overexpressed NS38 or N protein, green signals represent overexpressed LC3 (original magnification 63×; oil immersion objective). Scale bar, 5 μm. K The percentage of cells in which LC3 aggregates (J) was quantified under a 20× immersion objective (SP8; Leica) and data were expressed as mean ± SD, n = 3. Statistical analysis was performed by the Student's t-test. Asterisks indicate significant differences from control (^∗P < 0.05). M Autophagosome-like structures detection by transmission electron microscopy (TEM). EPC cells were seeded in 6-well plates overnight and transfected with indicated plasmids (1 μg each) for 24 h. The cells were then analyzed by TEM, enlarged section indicating autophagic vesicles. Scale bar, 2 or 0.4 μm.