Fig. 3: Ctr-infection inhibits membrane insertion and oligomerization of Bax in mitochondria.

A Following treatment of Ctr-infected Bak KO cells with staurosporine (1 µM, 5 h), mitochondria were isolated, and same amounts of total protein were treated with the cross-linker bismaleimidohexane (BMH). Samples were analyzed for Bax by Western blotting and Hsp60 was used as loading control. Cross-linking demonstrated the presence of increased amounts of Bax-dimers upon staurosporine-treatment in non-infected cells but substantially less on mitochondria from Ctr-infected cells. Data are representative of three independent experiments. B Mitochondria were isolated from HeLa cells after 24 h infection with Ctr and incubated at 30 °C for the different times indicated to assess the diffusion of Bax from mitochondria. Supernatant (S) and pellet (P) fractions were obtained by centrifugation, immunoblotted and analyzed for Bax, Bak, Mcl-1, Hsp60, and chlamydial Hsp60 (Ctr). Data are representative of three independent experiments. C Mitochondria from either non-infected or Ctr-infected, Bak-deficient HeLa cells were isolated and directly subjected to sodium carbonate treatment to assess membrane-integration of Bax or boiled in Laemmli-buffer (M) directly to measure total Bax levels. After extraction, supernatants (S₂) and pellets (P, not extracted, mostly integral membrane protein) were collected, and all samples were analyzed by Western blotting for membrane-bound proteins. Cytochrome c is a control for soluble protein released by the extraction treatment (detected only in supernatant) and VDACs is a control for membrane-bound proteins (detected mostly in pellet). Na₂CO₃, sodium carbonate extraction. Data are representative of three independent experiments. D Mitochondria were isolated as in (C) and incubated without or with recombinant tBid. Pellet and supernatant fractions were obtained by centrifugation, and supernatants (S₁) were used to analyze tBid-mediated release of cytochrome c. Pellet fractions were then extracted by sodium carbonate to isolate integral membrane proteins (P) from extractable membrane proteins (S₂). All protein fractions were run on SDS-PAGE and analyzed for indicated proteins. Data are representative of three independent experiments. E HCT116 cells deficient in Bax and Bak and stably expressing GFP-BAX were infected with Ctr or kept non-infected for 26 h, and then apoptosis was induced by staurosporine. Retrotranslocation of Bax was measured at all different conditions. Note, the unchanged rapid diffusion of Bax from mitochondria of Ctr-infected cells upon apoptosis induction. Data are representative of three different measurements. P-values according to one way Anova.