The HN protein of Newcastle disease virus induces cell apoptosis through the induction of lysosomal membrane permeabilization
Fig 2
NDV infection induces the leakage of CTSB and CTSD from the lysosomal lumen to the cytoplasm in HeLa cells.
(A) HeLa cells infected with Herts/33 at 0.01 MOI for the indicated time points or mock-infected. Indicated proteins in both lysosomal and cytoplasmic fractions extracted from the cells were detected by Western blotting. Immature and mature types of cathepsins were indicated as “IM” and “M”, respectively. (B-E) The relative intensity of lysosomal CTSB (B) and lysosomal CTSD (D) or cytoplasmic CTSB (C) and cytoplasmic CTSD (E) were quantified by ImageJ software. LAMP3 and α-Tubulin were used as normalized controls for lysosomal and cytoplasmic fractions, respectively. (F&G) HeLa cells were infected with Herts/33 at 0.01 MOI for the indicated time points or mock-infected. The activity of cytoplasmic CTSB (F) and CTSD (G) was measured as described in Materials and Methods. The obtained fluorescence intensity was normalized to the concentration of individual protein. (H&K) HeLa cells were infected with Herts/33 at 0.01 MOI for indicated timepoints or mock-infected. The colocalizations of CTSB (H) or CTSD (K) with LAMP3 were observed using confocal microscopy after immunostaining the cells with indicated antibodies. Scale bars, 20 μm. (I&J) Manders’ Colocalization Coefficients of CTSB (I) or CTSD (J) with LAMP3 were quantified by ImageJ software. Error bars represent SDs for triplicate analyses of three independent experiments (B-F) or SDs for 15 cells (I&J). All significance analyses were assessed using one-way ANOVA with Dunnett’s multiple comparisons test.