2 with shaking (160 rpm) at 42°C under microaerobic condition. Fifteen mL aliquots of NTCT 11168 culture (in triplicates) were treated with either sham (ethanol solvent for Ery), an inhibitory dose of Ery (4 mg/L; 16× MIC), or a sub-inhibitory dose of Ery (0.125 mg/L; 0.5× MIC). All cultures including the sham control
were thoroughly mixed and statically incubated under microaerobic conditions for 30 minutes at 42°C. Strain JL272 was treated with 4 mg/L Ery (16× MIC of the wild-type strain) or the sham under the same condition as with NCTC 11168. After 30 minutes treatment, the cultures were immediately mixed with RNAprotect™ (Qiagen, Valencia, CA) to stabilize the total bacterial RNA.
Total RNA was extracted using the RNeasy Mini kit (Qiagen) according to the manufacturer’s protocol and treated with TURBO DNase (Invitrogen, Carlsbad, learn more CA). RNA quantity was determined by OD260 reading using a NanoDrop spectrometer (Thermo Scientific, Wilmington, DE), and the purity was assessed by denaturing agarose gel electrophoresis. RNA samples confirmed free of DNA contamination by PCR of 16S rRNA gene, were stored at −80°C until use. Three independent MAPK Inhibitor Library screening RNA isolations (biological replicates) were performed for microarray experiments. C. jejuni microarray slides (version 3 for NCTC 11168 inhibitory treatment, version 4 for NCTC 11168 sub-inhibitory treatment, and version 1 for JL272 Ery treament) were designed and provided by the Pathogen Functional Genomics
Resource Center (PFGRC) at the J. Craig Venter Institute (JCVI, Rockville, MD). cDNA synthesis, labeling of cDNA and hybridization of labeled cDNA to the microarray slides were performed according to the JCVI’s protocol (http://pfgrc.jcvi.org/index.php/microarray/protocols.html ). For each pair of treated and untreated samples, hybridizations were performed with RNA samples prepared from three independent experiments, with the cDNA HDAC inhibitor alternately labeled with Cy3 and Cy5 for the pair in each slide. Slides were dried using a microarray high speed centrifuge (Arrayit, Sunnyvale, CA) and immediately scanned at a wavelength of 550 nm for Cy3 and 650 nm for Cy5 using a General Progesterone Scanning ScanArray 5000 (PerkinElmer, Boston, MA) at 10 μm resolution. Slide information and annotation files were obtained from the JCVI website (http://pfgrc.jcvi.org/index.php/microarray/available_microarrays/.html). The fluorescence intensities were collected and converted to digital signal by ImaGene software (BioDiscovery, EI Segundo, CA). The fluorescence intensity values were logarithm-transformed, median background corrected, and LOWESS normalized. The normalized gene expression data were analyzed using moderated-t test implemented in the R package, LIMMA . In this study, a p-value < 0.