Figure 3.

Distribution of Chlamydia CT775 is altered by LDs formation. HeLa cells were grown on coverslips and transfected with the GFP-CT775 construct for ∽44 h (A). Cells were infected with C. trachomatis strain D 4–6 h before transfection (B and D). Lipid droplets formation was induced by addition of 100 μmol/L of oleic acid 24 h after transfection (C and D). LDs were labeled in vivo by BODIPY C12 (red) for 1 h (C). BODIPY was chased for 30 min and cells were fixed for 30 min in 4% paraformaldehyde made in PBS. Bacteria were detected by staining the Chlamydia chromosome with the DNA dye Hoechst 33258 (blue). Images were taken with a Zeiss LSM710 confocal microscope equipped with 63× objective. Nuclei and inclusion are indicated with N and Inc, respectively. Formation of LDs drastically changed distribution of the CT775 tagged protein (A and B vs. C) and quantification analysis determined that 76% of the GFP-CT775 signal co-localized with the BODIPY signal with a tMCC of 0.8481 in (C). (D) Western-blot detection of the GFP-CT775 (58 kDa), GFP-ACBD6 (61 kDa) and of the LDs marker ADRP. Protein extract of HeLa cells transfected with the GFP-CT775 construct (lane 1), of LDs purified from GFP-transfected cells (lane 2), and of LDs purified from GFP-CT775 transfected cells (lane 3) were separated on 10% SDS-PAGE and probed with an anti-GFP antibody (G1544; Sigma). The same antibody was used to detect GFP-ACBD6 in LDs obtained from GFP-transfected cells (lane 1), in protein extract of cells transfected with the GFP-ACBD6 construct (lane 2), and in LDs purified from Chlamydia-infected GFP-ACBD6 transfected cells (lane 3). The LDs marker ADRP was detected with an antibody (PA5-25042; ThermoFisher Scientific) in the three LDs samples isolated from GFP-transfected cells (lane 1), from cells transfected with GFP-CT775 (lane 2), and from infected cells transfected with GFP-ACBD6 (lane 3). LDs, lipid droplets; tMCC, thresholded Manders’ colocalization coefficients.