L; incubated on ice for 1 h; Sigma), deoxycholate (2.eight mg/ml; incubated at 37 for 20 min; Fisher Scientific, Pittsburgh, PA), and DNase (4.five g/ml; incubated at room temperature for ten min; Roche, Branchburg, NJ) in lysis buffer (50 mM Tris, 100 mM NaCl, pH 7.five) supplemented with protease inhibitor (Total EDTA-free cocktail tablets, Roche); and disrupted by sonication using a model 505 sonic dismembrator (four 30-s pulses at 40 amplitude having a 30-s pause in between pulses; Fisher Scientific). Lcn2-GST was purified in the lysate applying a glutathione Sepharose 4B bead column (GE Amersham, Piscataway, NJ) followed by elution with glutathione elution buffer (50 mM Tris, 40 mM reduced glutathione [Sigma], pH 8.5) and overnight cleavage employing human thrombin (25 U per liter of E. coli; Sigma) through dialysis by way of a 10,000-MWCO membrane (Thermo Fisher Scientific) in buffered remedy (50 mM Tris, 100 mM NaCl, pH 7.5). Digested protein then was sterilized employing a 0.22- m filter (EMD Dipeptidyl Peptidase Inhibitor Species Millipore) and gel filtered working with a Superdex 75 column attached to an AKTA fast-performance liquid chromatography (FPLC) program (GE Healthcare) using buffer containing phosphate-buffered saline (PBS) to remove GST. The biological activity of purified Lcn2 was confirmed by retention with Fe-Ent after centrifugation over a 10,000-MWCO column as measured by absorbance at 340 nm and development inhibition of lipocalin-sensitive K. pneumoniae strain KP20 when added to human serum, as previously described (13). CAS assay. The chrome azurol S (CAS) assay was performed to identify the iron-chelating capabilities of Ent, GlyEnt (salmochelin S4), and Ybt at concentrations between 1 and 200 M as previously described (28). Microarray analysis. A549 cells have been stimulated overnight as described above. RNA was purified applying the miRNeasy kit (Qiagen) and submitted to the University of Pennsylvania microarray facility for hybridization on the Procollagen C Proteinase Species Affymetrix human gene 1.0ST gene chip (University of Pennsylvania microarray facility). Transcript abundance was estimated with the robust multiarray typical (RMA) algorithm and log transformed (29). A cutoff to get a important distinction in gene expression among ex-September 2014 Volume 82 Numberiai.asm.orgHolden et al.perimental groups of a fold change of 1.three having a P value of 0.01 was used. Gene sets with substantial modifications were used for enrichment evaluation by comparison for the Broad Institute Molecular Signatures Database (http: //broadinstitute.org/gsea/msigdb/index.jsp) (30). Gene ontology terms for every gene had been obtained by way of downloads of annotation files from the Affymetrix website. Calcein therapy. A549 lung epithelial cells have been seeded and serum starved as described above. Cells were washed twice with RPMI without having phenol red (Invitrogen) and pretreated with 1 M calcein (Sigma) for 30 min in a standard cell culture incubator. Cells then have been washed twice with RPMI with out phenol red and treated overnight with siderophores with or with out FAC. Fluorescence imaging was performed with an Olympus IX52 inverted microscope (Center Valley, PA), and photos had been analyzed with cellSens Entry imaging computer software (Olympus). Western blotting. A549 lung epithelial cells have been seeded, serum starved, and stimulated as stated above. Following overnight stimulation, cellular fractionation was performed to gather nuclear proteins as previously described (31) or with radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris-HCl, pH 7.five, 150 mM NaCl.
