Figure 2.

Activation of Bax and Bak by UV light is inhibited in C. trachomatis–infected cells. (A) On the left, detection of active Bax by flow cytometry is shown. HeLa cells were either infected or left uninfected. At the time points indicated, cells were treated with UV light (1,000 J/m²; bottom) or not (top). Cells were collected 6 h later and stained for active Bax. Normal line/shaded area, uninfected cells; bold line, C. trachomatis–infected cells. On the right, detection of active Bak is shown. Data are representative of three experiments. (B) Detection of Bax and Bak by Western blot. HeLa cells were infected or left uninfected. 24 or 48 h later, cells were collected and subjected to Western blotting with antibodies specific for total Bax, total Bak, chlamydial HSP60 (as a control of infection), and tubulin (as a loading control). Similar results were obtained in three independent experiments. (C) Active Bax localizes to mitochondria and is only detectable in noninfected cells. In Hep2 cells with or without (mock) chlamydial infection, apoptosis was induced by UV irradiation (1,000 J/m²). After 6 h, cells were doubly labeled with anti-active Bax (red) and the mitochondrial marker MitoTracker Green FM (left, green; colocalization results in yellow color) or anti-active Bax (green) and anti-chlamydial LPS (right, red). Active Bax was not detected in infected cells (arrows). Asterisks indicate noninfected cells positive for active Bax. For the results shown in the right panel, infectious dose was reduced (MOI: 0.5). Similar results were obtained in three independent experiments.