Figure 1

In vivo labeling of lipids in Chlamydia-infected cells. (A) HeLa cells were grown in T-75 flasks and were infected with C.t. or maintained uninfected. After 24 to 30 hours of infection, the cells were labeled by addition of 5 µM C₁-BODIPY500/510 C₁₂ to the medium, After incubation at 37 °C for an additional 2 hours, the cells were washed, and fresh medium containing triacsin C (10 µM), rosiglitazone G (100 µM) or ethanol (untreated cells) was added. After 1 hour of incubation at 37 °C, 10 µM ¹⁴C-C₁₆-OH complexed with BSA was added to the medium. The cells were washed and harvested after a further 1-hour incubation at 37 °C (see Methods), and lipids and proteins were extracted. (A) Lipids were separated by thin-layer chromatography, and dually labeled lipids were visualized with a FluoChem camera and after exposure to a phosphorimager screen scanned with a StormImager. A sample obtained from HeLa-infected cells (HeLa/Ct) is shown in lane 1 of the TLC plate. C₁-BODIPY500/510 C₁₂ (lane 2) and ¹⁴C-C₁₆-OH (lane 3) were used as migration standards. The migration positions of FA and phospholipids are indicated on the left. (B) Proteins were separated by SDS-PAGE, transferred to a PVDF membrane and blotted with monoclonal antibodies against C.t. HSP60 (upper panels) and human GAPDH (lower panel).