C. pneumoniae disrupts eNOS trafficking and impairs NO production in human aortic endothelial cells
Article first published online: 9 SEP 2014
© 2014 John Wiley & Sons Ltd
Volume 17, Issue 1, pages 119–130, January 2015
How to Cite
Mueller, K. E. and Wolf, K. (2015), C. pneumoniae disrupts eNOS trafficking and impairs NO production in human aortic endothelial cells. Cellular Microbiology, 17: 119–130. doi: 10.1111/cmi.12341
- Issue published online: 18 DEC 2014
- Article first published online: 9 SEP 2014
- Accepted manuscript online: 11 AUG 2014 04:53AM EST
- Manuscript Accepted: 5 AUG 2014
- Manuscript Revised: 1 AUG 2014
- Manuscript Received: 18 MAR 2014
- American Heart Association. Grant Number: 11SDG5580014
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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