biostudies-arrayexpressUnknownTranscriptomicsGenomicsProteomicsJoerg Mostertztranscription profiling by arrayHomo sapiensHomo sapienshttps://www.ebi.ac.uk/biostudies/studies/E-GEOD-65018Responsiveness of cells to alpha-toxin (Hla) from Staphylococcus aureus appears to occur in a cell-type dependent manner. Here, we compare two human bronchial epithelial cell lines, i.e. Hla-susceptible 16HBE14o- and Hla-resistant S9 cells, by a quantitative multi-omics strategy for a better understanding of Hla-induced cellular programs. Phosphoproteomics revealed a substantial impact on phosphorylation-dependent signaling in both cell models and highlights alterations in signaling pathways associated with cell-cell and cell-matrix contacts as well as the actin cytoskeleton as key features of early rHla-induced effects. Along comparable changes in down-stream activity of major protein kinases significant differences between both models were found upon rHla-treatment including activation of EGFR and MAPK1/3 signaling in S9 and repression in 16HBE14o- cells. System-wide transcript and protein expression profiling indicate induction of an immediate early response in either model. In addition, EGFR and MAPK1/3-mediated changes in gene expression suggest cellular recovery and survival in S9 cells but cell death in 16HBE14o- cells. Strikingly, inhibition of the EGFR sensitized S9 cells to Hla indicating that the cellular capacity of activation of the EGFR is a major protective determinant against Hla-mediated cytotoxic effects. Design includes two different human bronchial epithelial cell lines, one control treatment (mock treatment for 2 hours) and one condition (2 alpha toxin treatment for 2 hours). Experiments for both cell line were replicated twice (biological replication).biostudies-arrayexpressLabeling - For each sample, 200 ng of total RNA was reverse transcribed into cDNA, amplified, and in vitro transcribed to cRNA. Sense-strand cDNA was generated from 10 µg of purified cRNA using random primers, followed by fragmentation and labelling using 5.5 µg of purified sense-strand DNA.Scaning - Scanning was performed by GeneChip Scanner 3000 7G (Affymetrix); raw CEL files were generated using the GCOS software. Quality assessment of all hybridizations was carried out by inspecting scan images and by reviewing external and endogenous controls using the Expression Console software (Affymetrix).Nucleic Acid Extraction - Total RNA was isolated using the TRIzol reagent (Invitrogen). RNA was purified using the RNA Clean-Up and Concentration Micro Kit (Norgen) and concentrations were measured using a ND-1000 spectrophotometer (Thermo Fisher Scientific Inc). RNA integrity was validated by means of the lab-on-chip capillary electrophoresis technology (Bioanalyzer 2100, Agilent Technologies). Only RNA samples with an RNA integrity number (RIN)>9.5 [34], 260/280 nm≥1.8, 260/230 nm≥1.9 were used for microarray analyses.Hybridization - Biotinylated sense-strand DNA was then hybridized onto the Affymetrix GeneChip Human Gene 1.0 ST arrays for 16 h. Arrays were washed and stained using the Fluidics Station 450.MIAME ScoreRaw DataOrganizationAssays and DataProcessed DataMAGE-TAB FilesArray DesignsJoerg MostertzRavi ChilukotiData Transformation - Data analysis was carried out using Rosetta Resolver system for gene expression data analysis (Rosetta Bio software). In brief, the raw signals of the gene-specific probes were summarized using the Robust Multi-array Average algorithm and data transformation for array comparability was achieved by performing quantile normalization. Genes exhibiting significantly different expression on the RNA level were identified using the following cut-off criteria: one-way analysis of variance with subsequent Benjamini and Hochberg false discovery rate multiple-testing correction on pair-wise comparisons (ANOVA, p≤0.05), signal correction statistics (Ratio Builder, p≤0.05) and fold-change≥1.5-fold. Probe-set transformation into genes was performed by using the Rosetta Resolver transformation tool based on the Entrez Genes/Unigenes search engine (NCBI). processed_data.txt: Columns A to C: Gene Identification; Columns D to J: Summarized Intensity signals; Column L & O: Fold Change, Column M,N and P, Q: p-value and post hoc p-value, respectively ID_REF = VALUE = RMAfalseThe epithelial cell transcriptome after alpha-toxin treatmentResponsiveness of cells to alpha-toxin (Hla) from Staphylococcus aureus appears to occur in a cell-type dependent manner. Here, we compare two human bronchial epithelial cell lines, i.e. Hla-susceptible 16HBE14o- and Hla-resistant S9 cells, by a quantitative multi-omics strategy for a better understanding of Hla-induced cellular programs. Phosphoproteomics revealed a substantial impact on phosphorylation-dependent signaling in both cell models and highlights alterations in signaling pathways associated with cell-cell and cell-matrix contacts as well as the actin cytoskeleton as key features of early rHla-induced effects. Along comparable changes in down-stream activity of major protein kinases significant differences between both models were found upon rHla-treatment including activation of EGFR and MAPK1/3 signaling in S9 and repression in 16HBE14o- cells. System-wide transcript and protein expression profiling indicate induction of an immediate early response in either model. In addition, EGFR and MAPK1/3-mediated changes in gene expression suggest cellular recovery and survival in S9 cells but cell death in 16HBE14o- cells. Strikingly, inhibition of the EGFR sensitized S9 cells to Hla indicating that the cellular capacity of activation of the EGFR is a major protective determinant against Hla-mediated cytotoxic effects. Design includes two different human bronchial epithelial cell lines, one control treatment (mock treatment for 2 hours) and one condition (2 alpha toxin treatment for 2 hours). Experiments for both cell line were replicated twice (biological replication).2015-03-31T00:00:00Z2023-09-11T22:05:37.32Z2022-03-09T02:12:10.308ZE-GEOD-65018GSE65018EFO_0002768