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.

Viruses 2015, 7, page-page three compounds ( Figure 1A ), but at varying degrees, which was also dependent on the application time point. NIM811 was the least effective compound, while the effectiveness of PFTμ was comparable to z-VAD-fmk. The greatest level of reduction in the floater population was reached after application of PFTμ (27% ± 2% of the SC) and z-VAD-fmk (30% ± 3% and 28% ± 3% of the SC for 12.5 μM and 25 μM, respectively) at 24 hpi. The respective SC (DMSO)-treated sample was set at 100%; (B) RV titer was determined by plaque assay for supernatants collected at 3 dpi after drug application at the time point with maximal efficacy (NIM811 at 2 hpi, z-VAD-FMK and PFTμ at 24 hpi). PFT α was added at 2 hpi. In comparison to the SC, changes in viral titer were not significant; (C) Characterization of the presence of an apoptotic laddering in the floater population collected from the supernatant of the SC-and NIM811 (2 hpi)-and PFTμ (24 hpi)-treated and RV-infected Vero cells. Floaters were collected at 3 dpi and then counted before the same number of floaters (1 × 10 6 ) was subjected to DNA extraction and analysis by agarose gel electrophoresis. As a control, mockand staurosporine (5 μM)-treated Vero cells are shown in comparison to the floater population (3 dpi) of RV-infected cells. M, molecular size marker; (D) Time course analysis (1, 2, 3 dpi) of the activation of caspase 3 and 7 in RV-infected Vero cells; (E) Analysis of necrotic (PI-positive) and early (annexin V-EGFP-positive) and late-apoptotic events (annexin V-enhanced green fluorescent protein (EGFP) and PI-positive) by annexin V-EGFP and PI staining by microscopic analysis of living cells; (F) Determination of caspase 3 and 7 activity after application of PFTμ and NIM811 in comparison to the SC. SC, solvent control; scale bar, 20 μm. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.