Fig. 8: OmpA-expression inhibits mitochondrial apoptosis in HeLa cells, with OmpA inserting into mitochondrial membranes and blocking the tBid-induced release of cytochrome c. | Cell Death & Differentiation

Fig. 8: OmpA-expression inhibits mitochondrial apoptosis in HeLa cells, with OmpA inserting into mitochondrial membranes and blocking the tBid-induced release of cytochrome c.

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

Fig. 8

A HeLa cells carrying a construct encoding either OmpA (Tet OmpA) or GFP (Tet GFP) under the control of a tetracycline-inducible promoter were treated with anhydrotetracycline hydrochloride (AHT) to induce OmpA or GFP for 48 h. To analyze the subcellular localization of OmpA, cells were fractionated and mitochondrial and cytosolic fractions were subjected to Western blotting. Bak and VDAC were used as mitochondrial markers. B Mitochondria isolated from HeLa cells where OmpA or GFP had been induced for 48 h with AHT were incubated with recombinant tBid for 30 min as indicated. Mitochondria were pelleted, and supernatant fractions (labelled S1) were stored to assess tBid-induced release of cytochrome c. Mitochondrial pellets were resuspended in sodium carbonate buffer (pH 11.5) for protein-extraction. Membranes were then pelleted again (pellet fractions from this step are labelled P). Supernatants from this step (S2) were precipitated with trichloroacetic acid. Fractions were analyzed by Western blotting for cytochrome c (cyto c), OmpA, mitochondrial Hsp60, tBid, and VDAC. Note the retention of OmpA in the membrane (pellet) fraction. Less cytochrome c was released from OmpA-containing mitochondria compared to control (S1; no cytochrome c was seen in the precipitated S2-fractions, presumably as a matter of sensitivity). Data are representative of three independent experiments. C Mitochondria isolated from HeLa cells in which GFP or OmpA had been induced as above were incubated with various concentrations of recombinant tBid. Pellets (P) and supernatants (S) were collected and analyzed by Western blotting. Hsp60 was used to identify mitochondria. Note the reduced release of cytochrome c from OmpA-expressing mitochondria. Data are representative of three independent experiments. D Transgenic HeLa cells constitutively expressing GFP or OmpA from Ctr, were treated with ABT-737 (1 µM) and S63845 (500 nM) for 4 h. Cells were fixed, stained for active caspase-3 and analyzed by flow cytometry. Data show means/SEM of six independent experiments. ****p < 0.0001 two way anova multiple comparisons.

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