The HN protein of Newcastle disease virus induces cell apoptosis through the induction of lysosomal membrane permeabilization
Fig 7
The viral HN protein contributes to NDV-induced LMP and apoptosis.
(A) Western blotting analysis of LAMP1 and LAMP2 protein levels in HeLa cells after transfection with plasmids expressing indicated viral proteins for 48h. (B&C) Western blotting analysis of LAMP1 and LAMP2 protein levels in HeLa cells after transfection with indicated doses of plasmids expressing HN (B) or F (C) protein for 48h. (D) Confocal microscopy images of LMP level in HeLa cells using anti-galectin 3 (green) and anti-LAMP1 (red) antibodies after transfection with indicated plasmids for 48h. Scale bars, 20 μm. (E&F) The interaction between HN and LAMP1 (E) or LAMP2 (F) was detected by an immunoprecipitation assay with anti-flag or control IgG antibodies after transfection with indicated plasmids into HeLa cells for 48h. (G&H) Confocal microscopy images of the interaction between HN and LAMP1 (G) or LAMP2 (H) using anti-HN, anti-LAMP1, or anti-LAMP2 antibodies after infection of HeLa cells with Herts/33 at 0.01 MOI or mock-infected for 24h. Scale bars, 20 μm. (K) HeLa cells were transfected with siRNAs targeting LAMP1 or LAMP2 or plasmids expressing LAMP1 or LAMP2 for 48h. Then LAMP1 or LAMP2 protein levels were detected by Western blotting. The red boxes indicate the siRNA selected for this study. (I&J) HeLa cells were transfected with plasmids expressing LAMP1 or LAMP2 (M) or siRNAs targeting LAMP1 or LAMP2 (N) for 48h. Then the LMP level was observed by confocal microscopy using anti-galectin 3 (green) and anti-LAMP1 (red) antibodies following transfection with plasmid expressing HN protein for another 48h. Scale bars, 20 μm. (L&M) Manders’ Colocalization Coefficients of galectin 3 with LAMP1 are quantified by ImageJ software and shown in (L) for (I) and (M) for (J). (N&O) HeLa cells were transfected with plasmids expressing LAMP1 or LAMP2 (N) or siRNAs targeting LAMP1 or LAMP2 (O) for 48h. Then the apoptosis rate was measured by AnnexinV-FITC/PI staining using flow cytometry following transfection with plasmid expressing HN protein for 48h. Error bars are SDs for 15 cells (L&M), or SDs for a triplicate analysis of three independent experiments (N&O). All significance analyses were assessed using One-way ANOVA with Dunnett’s multiple comparisons test.