The HN protein of Newcastle disease virus induces cell apoptosis through the induction of lysosomal membrane permeabilization
Fig 6
NDV infection induces deglycosylation and degradation of LAMP1 and LAMP2.
(A&B) HeLa cells infected with Herts/33 at 0.01 MOI for indicated timepoints. The mRNA levels (A) and fluorescence images (B) of LAMP1 and LAMP2 were measured by RT-qPCR or observed by confocal microscopy, respectively. The mRNA levels of LAMP1 and LAMP2 were normalized to GAPDH using the 2−ΔΔCt method. Scale bars, 20 μm. (C-E) HeLa cells infected with Herts/33 at 0.01 MOI for indicated time points or infected with Herts/33 at indicated MOIs for 24h. The protein levels of LAMP1 and LAMP2 (C) or LAMP3, LIMPII, and LAPTM5 (E) were assessed by Western blotting. The deglycosylated LAMP1 and LAMP2 in PNGase F-treated cell lysates were analyzed by Western blotting (E). (F-H) HeLa cells were infected with Herts/33 at 0.01 MOI for 12h or mock-infected. The remaining amounts of LAMP1 and LAMP2 after treatment with CHX (20 μg/mL) for indicated time points were measured using Western blotting (F). The remaining amount of LAMP1 (G) and LAMP2 (H) was calculated as the fold change of the protein present at 0h. (I-K) NDV-infected (0.01 MOI, 6h) HeLa cells were co-treated with CHX (20 μg/mL) and NH4CL (50 mM), or Ca-074 (10 μM), or Pep A (10 μM), or 3-MA (5mM), or MG132 (5 μM) or mock-treated for 12h. LAMP1 and LAMP2 protein levels in cell lysates were analyzed by Western blotting (I). The remaining amounts of LAMP1 (J) and LAMP2 (K) were calculated as the fold change of the protein to mock-treated cells at 0h. All quantitative data represent means ± SD (n = 3). Significance was assessed using Two-way ANOVA with Dunnett’s multiple comparisons test (A), or unpaired two-tailed Student’s t-test (G&H), or One-way ANOVA with Dunnett’s multiple comparisons test (J&K).