Evolutionary Conservation of Infection-Induced Cell Death Inhibition among Chlamydiales
Figure 4
Bax/Bak activation and cytochrome c release.
(A) Immunofluorescence staining of the effect of Simkania infection on cytochrome c distribution. HeLa cells with or without Simkania infection (MOI 1, 3 days) were treated with 20 ng/ml TNF-α+3 µg/ml Chx or carrier for 4 hours before analysis. Cells were stained for cytochrome c (green), Simkania/nuclei (blue) and viewed with a confocal microscope. Upon apoptosis induction cytochrome c is released from the mitochondria (uninfected+TNF-α), this was not the case for infected cells (infected+TNF-α). The infected cells are marked by white asterisk, n = 2 (B) Western blot analysis for cytochrome c release. Cytosol and mitochondria were isolated as described in the experimental procedures section and cytochrome c was detected by Western blot. Cytochrome c release was completely blocked by Simkania infection. Tom40 was used as a marker for mitochondria and Actin as a marker for cytosol. (C) Immunofluorescence staining of active Bax with a conformation sensitive antibody [65] after Simkania infection. HeLa cells were treated as described in Figure 4A. Images show mitochondrial localization of active Bax (green) in control-induced cells. Bax was not activated in TNF treated Simkania infected cells. The infected cells are marked by white asterisk. n = 2 (D) Immunoblot analysis of Bax and Bak after mitochondrial isolation and in vitro cross linking. HeLa cells with or without Simkania infection (MOI 1, 3 days) were treated with 20 ng/ml TNF-α+3 µg/ml Chx or carrier for 4 hours before mitochondria isolation, in vitro cross linking and analysis. Bax and Bak (black arrows) are not activated in Simkania infected induced cells as shown by background Bax and Bak oligomerisation (brackets). Equal loading was verified by Bradford assay and Tom40 western blot. Input Bak, Bax and Actin showed no differences in expression or loading. n = 2.