Fig. 9: Ectopic expression of OmpA inhibits Bak-activation at the same step as Ctr-infection. | Cell Death & Differentiation

Fig. 9: Ectopic expression of OmpA inhibits Bak-activation at the same step as Ctr-infection.

From: Chlamydia trachomatis inhibits apoptosis in infected cells by targeting the pro-apoptotic proteins Bax and Bak

Fig. 9

A Mitochondrial fractions from control or OmpA-expressing cells were incubated without or with trypsin to identify the enhanced Bak-protease sensitivity upon its tBid-dependent activation. Intact Bak or OmpA with their cleavage products were detected by immunoblotting. To assess cytochrome c release supernatants were analyzed separately using a cytochrome c antibody. Note the trypsin sensitivity of OmpA upon tBid-treatment of mitochondria. GFP or OmpA was induced by treatment with anhydrotetracycline for 48 h. (representative of three independent experiments). B Mitochondria isolated from uninfected, FLAG-OmpA- or GFP-expressing HeLa cells were incubated with various concentrations of tBid for 30 min. Mitochondrial pellets were lysed and analyzed by BN-PAGE, followed by Western blotting for Bak, OmpA, and VDAC. Reaction supernatants were analyzed separately by SDS-PAGE to control for cytochrome c-release. Bak-containing complexes were visualized using antibody Bak(aa23-38); (see Supplementary Table S1). OmpA- and VDAC-containing complexes were detected using specific antibodies. The arrowhead indicates Bak-dimers. Data are representative of three independent experiments. C HeLa cells carrying tetracycline-inducible constructs were treated with AHT for 48 h to induce GFP or (untagged) OmpA. Mitochondria were isolated and treated with tBid as indicated. In some reactions, proteins were crosslinked using the cysteine crosslinker BMH. To control for cytochrome c-release and its inhibition by OmpA expression, released fractions (Supernatant) were analyzed separately by Western blotting for cytochrome c. Results are representative of three independent experiments. D After treatment of HeLa cell variants (Wt, cells deficient in VDAC2, cells constitutively expressing FLAG-OmpA or cells deficient in VADC2 and expressing FLAG-OmpA) with the combination of 1 µM ABT-737 and 0.5 µM S63845 for various times, active caspase-3 was determined by flow cytometry analysis. Data show means/SEM of three independent experiments.

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