Figure 1

Activation of apoptosis by intrinsic and extrinsic pathways in BTV infected mammalian cells. A: Detection of cleavage of caspase-3 in BTV-1 infected HeLa cells at different times p.i. B: Immunofluorescence of BTV-1 infected HeLa cells shows the morphological changes associated with apoptosis. (a) Uninfected cells stained with Hoescht, (b) BTV-1 infected stained with Hoescht, the nuclear blebbing and condensation is indicated by a white arrow, (c) BTV-1 infected stained with Hoescht (blue) and immunofluorescence detection of VP5 (green) and (d) BTV-1 infected stained with Hoescht (blue), VP5 (green) and caspase-3 (red) detected by immunofluorescence. BTV infected HeLa cells undergoing apoptosis are indicated by the open arrow and uninfected cell indicated by the closed arrow. (e) Uninfected HeLa cells and (f) BTV-1 infected HeLa cells stained with Hoescht (blue) and anti-active caspase-8 monoclonal clonal antibody (red). Cleaved caspase-8 is visible in the BTV infected cells (open arrow) C: Detection of caspase-8 cleavage in BTV-1 infected HeLa cells at different time p.i. The procaspase-8 (43 kD), cleaved caspase-8 (18 kDa) product and a 26 kDa non-specific band (*) are indicated. D: Detection of cytochrome C (12 kDa) in the mitochondrial (top) and cytosolic (bottom) fractions of BTV-1 infected cells at different times post infection, demonstrating the release from the mitochondria and its accumulation in the cytosol. E: Western immunoblot analysis of caspase-9 cleavage in the whole cell lysate of BTV-1 infected cells. The procaspase-9 (47 kDa) and cleaved (37 kDa) products of caspase-9 are indicated.