The HN protein of Newcastle disease virus induces cell apoptosis through the induction of lysosomal membrane permeabilization
Fig 8
The sialidase activity of the HN protein mediates the deglycosylation and degradation of LAMP1 and LAMP2.
(A) Western blotting analysis of LAMP1 and LAMP2 deglycosylation and degradation in HeLa cells transfected with plasmid expressing HN protein and treated with Peramivir (30 μg/mL) or Zanamivir (20 μg/mL) at the indicated time points post-transfection. (B&C) HeLa cells were transfected with plasmids expressing HN protein or mock-transfected. Then the cells were treated with Peramivir (30 μg/mL) or Zanamivir (20 μg/mL) or DMSO or untreated for 48h. The LMP level was observed by confocal microscopy using anti-galectin 3 (green) and anti-LAMP1 (red) antibodies. Scale bars, 20 μm (B). Manders’ Colocalization Coefficients of galectin 3 with LAMP1 are quantified by ImageJ software and shown in (C). (G) Sialidase activity was measured in HeLa cells transfected with the indicated plasmids for 48h and is presented as relative change to the cells transfected with plasmids expressing wild-type HN. The red dotted line indicates the difference threshold. (F) Western blotting analysis of LAMP1 and LAMP2 deglycosylation and degradation in HeLa cells transfected with the indicated plasmids or mock-transfected for 48h. (D&E) The interaction between HN and LAMP1 (D) or LAMP2 (E) in HeLa cells was detected by an immunoprecipitation assay with anti-flag or control IgG antibodies after transfection with indicated plasmids for 48h. (H) The LMP level was observed by confocal microscopy using anti-galectin 3 (green) and anti-LAMP1 (red) antibodies following transfection with the indicated plasmids or mock-transfected for 48h. Scale bars, 20 μm. (I) Manders’ Colocalization Coefficients of galectin 3 with LAMP1 are calculated by ImageJ software. (J) The apoptosis rate was measured by AnnexinV-FITC/PI staining using flow cytometry following transfection with indicated plasmids or mock-transfection for 48h. Error bars are SDs for a triplicate analysis of three independent experiments (G&J), or SDs for 15 cells (C&H). All significance analyses were assessed using One-way ANOVA with Dunnett’s multiple comparisons test.