BH3-only sensors Bad, Noxa and Puma are Key Regulators of Tacaribe virus-induced Apoptosis
Fig 2
TCRV infection induces classical hallmarks of the intrinsic apoptotic pathway.
(A) Schematic model of key processes in intrinsic apoptosis. Canonical events associated with intrinsic apoptosis include mitochondrial disorganization and membrane disruption, leading to Cytochrome c (Cyt c) release, caspase (Casp) activation (especially Casp9 and Casp3), phosphatidylserine (PS) flipping, and finally cell death (B) Caspase activity. Vero76 cells were mock-infected or infected (MOI = 2) with TCRV/UV-inactivated virus for 1–4 days, as indicated. Treatment with CPT (10 μM) for 24 h served as a positive control (Ctrl ind.). Cell lysates were analysed by Western blot for initiator Casp8 and Casp9, as well as executioner Casp3, using antibodies detecting both the full-length form (Casp) and cleavage products (cCasp) of the respective proteins. Detection of viral NP served as a control for infection. (C) PS flipping. Cells infected as indicated above were stained with Annexin V-FITC and PI and analysed by flow cytometry. Representative dot-plots indicate the percentage of Annexin V positive (early apoptotic cells), PI positive (necrotic cells), and Annexin V/PI double-positive cells (late apoptotic and dead cells). Treatment with STS (1 μM) for 24 h served as a positive control (Ctrl ind.). Representative bright field images of cell morphology were taken each day and are shown with 100 μm scale bars. Quantifications of PS exposure are depicted as mean values and standard deviations representing data from two independent experiments. Statistical significance was determined using two-way ANOVA (**p≤0.01, ns not significant).