Azithromycin and ciprofloxacin have a chloroquine-like effect on respiratory epithelial cells

Vojo Deretic; Graham S Timmins

measurements. CF and normal cells, grown to post confluency, were incubated for 48 h with 1-100 µM AZT. Cells were fixed and stained with fluorescently labeled cholera toxin B for 1 h at room temperature. Relative fluorescence was calculated from actual grey levels of 10 random fields from 3 independent experiments. Postconfluent cells, treated with 1-100 µM AZT, were incubated with P. aeruginosa (PAO1) at a multiplicity of infection of 1:200 in MEM media without serum or antibiotic supplements. Samples were washed 3 times with PBS and lyses with 0.5% TX-100. Cell associated bacteria were plated on P. aeruginosa isolation agar and colonies counted. Treatment with Pseudomonas pro-inflammatory lipopeptide (lipotoxin) Lepta, NF-κB activation (luciferase assay), IL-8 and RANTES analysis were carried out as previously described 45, 66 . The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.03.29.008631 doi: bioRxiv preprint TGF-b production and furin activity analysis. TGF-β levels were determined using the PAI/L assay as described previously 67 . Furin enzyme activity was assayed in the presence of 0.25% Triton X-100 as a permeabilizing agent using boc-RVRRamc (100 µM) as a furin substrate. Fluorescence was measured at 380 nm excitation and 460 nm emission wavelengths. The inhibitory concentration of dec-RVKR-cmk (Calbiochem) for ExoA-mediated cell death, were assessed and 50 µM was employed in subsequent experiments.