Figure 1.
Changes of mitochondria in apoptotic cells induced with AfMNPV under confocal microscopy.
(A) Control cells stained with Mito-Tracker Green, showing mitochondrial normal distribution; (B) Infection of AfMNPV caused mitochondria to aggregate at 4 h post-infection; (C) Mito-Tracker Green staining showed a diffusion of fluorescence at 8 h post-infection, suggesting the disruption and depolarization of mitochondria. Bar = 10 µm.
Figure 2.
Transmission electron images of mitochondria in apoptotic cells induced with AfMNPV.
(A) Control cells showing mitochondria had normal ultrastructure with intact cristae and an elongated form; (B) AfMNPV infection for 4 h resulted in change of mitochondria that appeared swollen and rounded; (C) Mitochondria were further swollen and rounded, cristae of which disappeared in cells infected by AfMNPV for 8 h; (D) Mitochondria with swollen and vesicular-swollen morphology in cells infected with AfMNPV for 12 h. Bar = 500 nm.
Figure 3.
Cytochrome c localization in Sl-1 cells was altered during apoptosis.
Sl-1 cells infected with AfMNPV for 6 h were co-stained with anti -cytochrome c monoclonal antibody and Mito-Tracker red. After incubation with FITC-coupled secondary antibodies, cells were visualized by confocal laser scanning microscopy. Con: Control cells treated without AfMNPV; Inf: Cells infected with AfMNPV for 6 h.
Figure 4.
Cytochrome c stimulated caspase-3 activity in cell-free extracts.
Caspase-3 activity in the presence of cytochrome c was measured by a flourimetric assay using the substrate Ac-DEVD-AFC. C1: Control untreated with cytochrome c; 0–10 h: caspase-3 activity in samples treated with cytochrome c for different time points; C2: control treated without cytochrome c for 8 h.
Figure 5.
Cytochrome c dsRNA silenced cytochrome c expression in Sl-1 cells.
(A) Identification of recombinant plasmid pLitmus-cytc containing cytochrome c DNA fragment. pLitmus-cytc was digested with BamH I and EcoR I and then analyzed by agarose gel electrophoresis. M1: 1 kb maker, M2: 100 bp maker, S: plasmid DNA products. (B) Electrophoretic analysis of dsRNA transcribed in vitro. pLitmus-cyt c was digested with BamH I and EcoR I respectively and transcribed in vitro, and RNA products were electrophorised on agrose gel. M: 100 bp maker, S: RNA transcription products. (C) Cytochrome c mRNA level after treatment with dsRNA. Sl-1 cells treated with dsRNA for 0, 24, 48, and 72 h, and after inoculated with AfMNPV for 10 h, total RNA in each treatment was isolated. Cytochrome c mRNA was determined by semi-quantitative RT-PCR.
Figure 6.
Western blot analysis showed the decrease of abundance of cytochrome c after transfection of its dsRNA in Sl-1 cells.
(A) Sl-1 cells transfected with GFP dsRNA; (B) Sl-1 cells transfected with cyt-c dsRNA. M, protein marker. β-tubulin was used as an internal control. *: non-specific band.
Figure 7.
Effect of silencing of cytochrome c on apoptosis induced by AfMNPV in Sl-1 cells.
(A) Microscopy image of cells treated with AfMNPV and cyt-c dsRNA. 1. Cells infected with AfMNPV alone for 10 h; 2. Cells infected with AfMNPV for 10 h after treatment with cyt c dsRNA for 24 h; 3. Cells infected with AfMNPV for 10 h after treatment with cyt-c dsRNA for 48 h.; 4. Cells infected with AfMNPV for 10 h after treatment with cyt-c dsRNA for 72 h. (B) Flow cytometric analysis. Sl-1 cells treated with dsRNA for 48 h,and infected with AfMNPV. At 10 h post infection, cells were staining by PI and FITC-Annexin. Apoptosis was analyzed by flow cytometry. (C) Cyt-c dsRNA resulted in the decrease of apoptosis induced by AfMNPV in Sl-1cells. Data were representive for three independent experiments. *, p<0.05.
Figure 8.
Down-regulation of cytochrome c resulted in the reduction of caspase 3 and caspase-9 activity in AfMNPV-induced Sl-1 cell.
(A) Sl-1 cells treated with dsRNA for different time points, apoptosis was induced with AfMNPV, and then caspase-3 activity was measured at 10 h post-infection.1. Control cells treated alone with AfMNPV; 2. Cells treated with GFP dsRNA and virus; 3. Cells without any treatment; 4. Cells treated with dsRNA, 24 h later, infected by AfMNPV for 10 h; 5. Cells treated alone with dsRNA for 24 h; 6. Cells treated with dsRNA, 48 h later, infected by AfMNPV for 10 h; 7. Cells treated alone with dsRNA for 48 h; 8. Cells treated with dsRNA, 72 h later, infected by AfMNPV for 10 h; 9. Cells treated alone with dsRNA for 72 h. (B) Caspase-9 activity in cytochrome c dsRNA-treated Sl-1 cells after infection with AfMNPV for 10 h, compared with control cells.1. Normal cells; 2. Cells infected with AfMNPV; 3. Cells infected with AfMNPV for 10 h after GFP dsRNA treatment for 48 h; 4. Cells infected with AfMNPV for 10 h after cyt c dsRNA treatment for 48 hr. *, p<0.05.
Figure 9.
Inhibitor of caspase-9 blocked the activation of pro-caspase-3 in AfMNPV-induced Sl-1 cells.
Con: non-infected cells; V: AfMNPV-infected cells; V+Z-LEHD-FMK: AfMNPV-infected cells cultured in complete medium supplemented with inhibitor of caspase-9 (Z-LEHD-FMK).
Figure 10.
Inhibition of the function of Apaf-1 blocked apoptosis.
(A) The treatment of FSBA resulted in the decrease of the percentage of apoptotic cells under the inductuion of AfMNPV. (B) The treatment of FSBA inhibited activation of pro-caspase-3 in AfMNPV-infected Sl-1 cells. *, There were significant differences.
Figure 11.
Pathway of apoptosis mediated by cytochrome c in AfMNPV-infected Sl-1 cells.
MPT: mitochondrial permeability transition.