Activation of the Mitochondrial Apoptotic Signaling Platform during Rubella Virus Infection

Claus, Claudia; Manssen, Lena; Hübner, Denise; Roßmark, Sarah; Bothe, Viktoria; Petzold, Alice; Große, Claudia; Reins, Mareen; Mankertz, Annette; Frey, Teryl K.; Liebert, Uwe G.

Induction of apoptotic cell death was monitored by the binding of annexin V-EGFP (Annexin V-EGFP Apoptosis Detection Kit from GenScript (Piscataway, NJ, USA) according to the manufacturer's instructions. Viable cells are not permeable for PI (5 µM), while induction of apoptosis leads to binding of annexin V due to exposure of phosphatidylserine. Activity of caspase 3 and 7 was determined using the CellEvent caspase-3/7 ReadyProbes reagent (Thermo Fisher Scientific, Braunschweig, Gemany) according to manufacturer's instructions. Briefly, Caspase-3/7 activity was determined by microscopic analysis of the fluorescent events after cleavage of the non-fluorescent DEVD peptide. As a DNA counterstain, cells were stained with Hoechst bisbenzimide 33342 (Thermo Fisher Scientific, Braunschweig, Gemany), (2 µM). Samples were analyzed by fluorescence microscopy using a FITC/TRITC filter. 4.6. Calcein Release Assay for Assessment of Mitochondrial Permeability Transition Pore (mPTP) Opening mPTP opening was assessed with the MitoProbe transition pore opening kit (Thermo Fisher Scientific Life Technologies). Mitochondrial calcein is lost after the opening of the mPTP. Hence, the mPTP opening was assessed through evaluation of the mitochondrial fluorescence signal of calcein after the quenching of its cytosolic portion with CoCl 2 . Briefly, medium was replaced with warm Hank's balanced salt solution containing calcium (HBSS/Ca) and loaded with calcein-acetoxymethyl ester (calcein-AM at 1.0 µM) and CoCl 2 (1.0 mM). After an incubation of 3 h at 37 ¥ C, cells were washed with HBSS and directly processed to fluorescence microscopic analysis. Opening of mPTP was induced through overnight incubation with H 2 O 2 (0.003%).