Modulating CFTR expression may limit SHS induced bacterial survival.

Abstract

<p>(A) Immunoblotting of total protein extracts from RAW264.7 cells treated with VRT-532 (10μM; overnight, a known CFTR corrector and potentiator) or untreated group (control), show slightly higher protein levels of CFTR and reduced NF-κB levels in the treatment group as compared to the controls. β-actin was used as a loading control (n = 3). (B) CFTR and NF- κB protein expression (in 3A) was normalized to β—actin using an Image-J software. The densitometry analysis verifies that VRT-532 treatment can decrease NF-κB protein expression (*p<0.05) as compared to untreated control. (C) RAW264.7 cells were seeded on a 24-well plate and treated overnight with VRT-532 (10μM). Next, these cells were infected with <i>PA01</i>-GFP (MOI 10) and/or treated with cigarette smoke extract (CSE; 10%; SHS model) for 150mins. Representative bright field (top) and fluorescent microscopy images (bottom) are shown (magnification 40X, n = 4, white bar = 20μm). (D) CSE treatment (in 3C) significantly (**p<0.01) inhibits bacterial phagocytosis, while VRT-532 is unable to restore SHS impaired phagocytosis. (E) In a separate experiment (as described in 3C), media (100μl) was collected, spread on agar plates, and then incubated overnight at 37°C. CFU counts of the extracellular bacteria (media) indicates that VRT-532 treatment can significantly (*p<0.05) decrease SHS induced bacterial survival. Note, this experiment was done in parallel with <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0121200#pone.0121200.g002" target="_blank">Fig. 2E</a> (left panel, Rutin Hydrate), hence control and CSE samples used are common.</p

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