Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling of mouse RAW 264.7 cells treated with the oxidant hypochlorous acid (HOCl) - dose response

ABSTRACT: Hypochlorous acid (HOCl) is a potent oxidant that is produced endogenously in mammalian tissue by phagocytes. Exogenous exposure to HOCl also can occur following inhalation of chlorine gas. HOCl has been implicated as a source of oxidative stress associated atherosclerosis and other diseases. The purpose of this study was to identify dose-dependent transitions in cellular response to hypochlorous acid (HOCl), with a focus on understanding how various cellular defense and stress dose-responses overlap. Experiment Overall Design: RAW 264.7 cells were grown to 80-90% confluency in DMEM supplemented with supplemented with 10% fetal bovine serum (FBS), 10 mM HEPES, 100 U penicillin/ml, and 100 Âµg streptomycin/ml. Cells were treated with 0, 0.14, 0.35, 0.7, 1.4, 2.1, 2.8 or 3.5 mM HOCl in medium for 6 hr. Total RNA was isolated from cells with TRIzol according to manufacturer's instructions and then subjected to cleanup using RNase-Free DNase Set and RNeasy Mini kit. The resultant DNA-free RNA was diluted in RNase-free H2O and quantified by Nanodrop at 260 nm. The quality of RNA samples was confirmed using RNA Nano Chips with Agilent 2100 Bioanalyzer. RNA samples were stored at â70 oC until use. Experiment Overall Design: From 5 micrograms of total RNA, cDNA was synthesized using a one-cycle cDNA synthesis kit (Affymetrix Corp., Santa Clara, CA). cDNA was transcribed to cRNA which was then biotin-labeled using GeneChip IVT labeling kit. Fifteen micrograms of labeled cRNA were then hybridized to an Affymetrix Mouse Genome 430 2.0 Array at 45Â°C for 16 hr. Biological cRNA replicates (n = 3) were each hybridized to an individual array. After being washed using the GeneChip Fluidics Station 450, arrays were scanned using a GeneChip 3000 scanner and intensity values were extracted from the CEL file using Array Assist software (Stratagene, La Jolla, CA). Experiment Overall Design:

ORGANISM(S): Mus musculus

SUBMITTER: Courtney Woods

PROVIDER: E-GEOD-15457 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Purpose: The aim of this study was to determine the transcriptional response of P. aeruginosa PA14 to HOCl and HOSCN stress, and determine genes dependent on RclR (PA14_07340) regulation. Methods: PA14 WT and ∆rclR exponential phase cultures were exposed to sub-lethal amounts of HOCl or HOSCN (2.2 or 0.8 mM, respectively), or untreated, and after 20 minutes of exposure, RNA was extracted and gene expression analysed by RNA-seq. Reads Per Kilobase per Million mapped reads (RPKM) were determined and the log2 fold change of the RPKM values of treated WT compared to untreated WT, or treated WT compared to treated ∆rclR was calculated. Genes with a log2 fold change of >1.5 were considered upregulated and genes with a log2 fold change of <-1.5 were considered downregulated. Results: In the WT strain, HOCl exposure resulted in upregulation of 231 genes and downregulation of 20 genes, and HOSCN exposure caused upregulation of 105 genes and downregulation of 16 genes. There was considerable overlap in the transcriptional response to HOCl and HOSCN, with 74 genes being induced in response to both oxidants. Genes upregulated by HOCl and HOSCN included those involved in sulphur and taurine transport, those encoding the type 3 secretion system, the MexEF-OprN drug efflux pump, and the putative peroxiredoxin RclX (PA14_07355). Comparison of the treated WT and treated ∆rclR strains revealed that in the presence of HOCl RclR upregulated 42 genes and downregulated 7 genes, and in the presence of HOSCN upregulated 132 genes and downregulated 213 genes. In the presence of both HOCl and HOSCN, RclR was required for upregulation of rclX as well as pyocyanin biosynthesis genes. In the presence of HOSCN, RclR downregulated a large number of genes, including those encoding chaperones. Conclusions: Our data have identified that the transcriptional response of P. aeruginosa to HOCl and HOSCN is multifaceted and appears to be linked to antibiotic resistance and the virulence factors pyocyanin and type 3 secretion. Determination of the regulon of the transcriptional regulator RclR after HOCl and HOSCN exposure suggests RclR has a role in coordinating the P. aeruginosa response to these oxidants.

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Transcription profiling of mouse RAW 264.7 cells treated with the oxidant hypochlorous acid (HOCl) - dose response

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