Fig. S1. HAECs support productive growth of C. pneumoniae. Endothelial cells were infected with C. pneumoniae, AR-39 at the moi of ∼ 0.5. A one-step growth curve shows more than 10-fold decrease in number of infectious bacteria during 20 h post infection (p.i.). The number of new infectious EBs increases by 48 h p.i., when the differentiation of RBs to EBs is initiated. Differentiation continues up to 72 h p.i.

Fig. S2. Colocalization of eNOS with C. pneumoniae inclusions in BEAS-2B. Human bronchial epithelial cells (BEAS-2B) were infected with C. pneumoniae for 64 h then fixed and stained with anti-eNOS-specific mAb. The arrows indicate chlamydial inclusions.

Fig. S3.C. pneumoniae associates with eNOS and not iNOS. Confocal micrographs of C. pneumoniae-infected HeLa cells transfected at 45 h p.i. with either 3xFLAG–eNOS or 3xFLAG–iNOS for additional 19 h. Cultures were fixed with PLP, permeabilized with 0.1% saponin and labelled with anti-FLAG mAb. A FLAG tagged eNOS revealed a rim-like staining around C. pneumoniae inclusion. D No significant colocalization of iNOS was detected with chlamydial inclusion. B and E C. pneumoniae inclusions were counterstained with species-specific anti-MOMP rabbit antiserum. C and F show composite images. The arrows indicate chlamydial inclusions. Scale bar = 10 μm.

Fig. S4. Comparison of upper fractions between uninfected and C. pneumoniae-infected cells. Mock and C. pneumoniae-infected HAECs at 64 h p.i. were harvested into 0.25 M sucrose buffer, Dounce homogenized, and clarified lysates were centrifuged through discontinuous sucrose gradient. Immunoblots were probed with anti-eNOS and anti-Hsp90 mAb.

Fig. S5. NO release in HAECs infected with C. pneumoniae at 38 h p.i. A Mock and C. pneumoniae-infected HAECs at 38 h p.i. were stimulated with VEGF for 1 h. NO release was measured in culture media by chemiluminescence. ∼ 30% decrease in NO release was detected with chlamydiae-infected cells when compared to uninfected HAECs. B Immunoblot of whole cell lysates of mock and C. pneumoniae-infected HAECs probed with anti-eNOS, anti-caveolin-1 and anti-Hsp90 mAb were used as a control for cell number in NO release experiment.

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