Figure 9.

Vimentin is dispensable for C. burnetii replication. (A–C) HeLa cells were transfected with 50 nM non-targeting (nt) or vimentin-targeting (vim) siRNA. After 24 h post-transfection, cells were infected with C. burnetii (WT) at a MOI of 50. (A) At the time points post-infection indicated, total cell lysates were separated by SDS-PAGE and analyzed by immunoblot analysis using antibodies against vimentin and actin as loading control. The immune blot is representative of four independent experiments. (B) 48 h post-infection, cells were fixed and stained with an anti-vimentin antibody (green). Nuclei and bacterial DNA were stained with DAPI (blue). Images are representative of two independent experiments. N: nucleus, C: C. burnetii-containing vacuole. (C) At the time-points indicated, cell lysates were prepared by osmotic lysis. Bacteria, isolated by differential centrifugation, were used for preparation of genomic DNA (gDNA). Absolute quantification of bacterial genomic equivalents was performed by quantitative real-time PCR with primers specific for genomic IS1111 sequences. gDNA, prepared from axenically-grown C. burnetii cultures served as standard for genomic equivalents. Error bars represent mean standard deviations of four independent experiments. **p < 0.01.