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.

On the contrary, overnight incubation with 0.003% H 2 O 2 induces profound localization of CytC outside of mitochondria ( Figure 4A ). In the case of RV-infected Vero cells (3 dpi), a minor but distinct portion of cells appear to have lost CytC from their mitochondria ( Figure 4A ). To further validate this observation, selective permeabilization of the plasma membrane with digitonin was performed. First, the lowest possible concentration of digitonin was determined, which gives a detectable fluorescence signal for a cytoplasmic protein, in this case alpha-tubulin. As a positive control, Figure 4B ,C illustrate the signal for the cytoplasmic protein alpha-tubulin and the mitochondrial protein CytC after 2% (w/v) paraformaldehyde (PFA) fixation and permeabilization with methanol. Figure 4B highlights that permeabilization with digitonin at 25 µg/mL after 2% (w/v) PFA fixation gives a sufficient signal for alpha-tubulin after application of its respective antibody. However, a remarkable signal loss occurs after application of digitonin at 1 µg/mL ( Figure 4B ). Hence, digitonin was applied at 25 µg/mL to verify CytC localization outside of mitochondria. As shown in Figure 4C , no CytC signal can be detected in mock-treated cells. However, overnight incubation with 0.003% H 2 O 2 produces a distinct CytC signal in the cytoplasm, which was also detectable for RV-infected cells (3 dpi). In summary, single cells within the RV-infected population were positive for CytC-signals in the cytoplasm, outside of mitochondria.