biostudies-arrayexpressUnknownTranscriptomicsGenomicsProteomicsDaniel Humphreystranscription profiling by arrayHomo sapiensHomo sapienshttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-15046Aim of the experiment: To examine changes in gene expression in HT1080 fibroblasts following treatment with purified recombinant wild-type typhoid toxin (WT-tox) from Salmonella Typhi relative to a mutant H160Q variant lacking DNase activity (HQ-tox), in the presence and absence of serum. The toxin causes DNA damage in both G0/G1 phase and G2/S phase of the cell cycle. Serum-starvation results in host cell cycle arrest in G0/G1 phase, meaning that DNA damage in G2/S is not permissive in the absence of serum. This experiment aimed to uncouple toxin-induced host transcriptomic responses to DNA damage in G0/G1 and G2/S phases of the host cell cycle. Experimental workflow: HT1080 fibroblasts were seeded one day before intoxication into T75 tissue culture flasks for a 30% confluency in DMEM with 1% PenStrep. Cells to be intoxicated in the presence of serum were supplemented with 10% foetal bovine serum (FBS), whereas no FBS was added for serum-starved cells. The next day, cells were incubated for 2h with WT-tox (5 ng/ml) or negative control HQ-tox (5 ng/ml) followed by three washes with PBS and a 48h chase in DMEM supplemented with 1% PenStrep and with or without 10% FBS as appropriate. After 48 h, cells were collected by trypsinisation. RNA was isolated and samples analysed using a human Clariom S assay (ThermoFisher Scientific, 902927). Analysis was performed with Transcriptome Analysis Console 4.0 software (Applied Biosystems, Thermo Fisher Scientific).biostudies-arrayexpressSample Collection - HT1080 fibroblasts were seeded one day before intoxication into T75 tissue culture flasks for a 30% confluency in DMEM with 1% PenStrep. Cells to be intoxicated in the presence of serum were supplemented with 10% foetal bovine serum (FBS), whereas no FBS was added for serum-starved cells. The next day, cells were incubated for 2h with WT-tox (5 ng/ml) or negative control HQ-tox (5 ng/ml) followed by three washes with PBS and a 48h chase in DMEM supplemented with 1% PenStrep and with or without 10% FBS as appropriate. After 48 h, cells were collected by trypsinisation.Labeling - cDNA was synthesised from 100ng RNA of total RNA using the GeneChip WT PLUS Reagent Kit. The cDNA was then fragmented and biotin-labelled using the GeneChip WT Terminal Labelling Kit according to the manufacturer's instructions (ThermoFisher)Scaning - Scanning was done in the Affymetrix® GeneChip Scanner 3000 7G with autoloader. The GeneChip Operating Software (GCOS) software (ThermoFisher) was used to acquire and feature extract the data.Nucleic Acid Extraction - Total RNA was extracted using the Qiagen RNeasy kit according to manufacturer's instructions.Hybridization - Hybridisation and washing was done using the GeneChip™ Hybridization, Wash, and Stain Kit (ThermoFisher). Hybridisation was to GeneChip Clariom S Arrays for 16h at 60rpm and 45C. Washing and staining was done on an Affymetrix Fluidics Station 450.MIAME ScoreRaw DataOrganizationAssays and DataProcessed DataMAGE-TAB FilesArray DesignsDaniel HumphreysData Transformation - Analysis was performed using Transcriptome Analysis Console 4.0 software (Applied Biosystems, ThermoFisher).falseTranscriptome of human HT1080 cells treated with purified typhoid toxin in the presence and absence of serumAim of the experiment: To examine changes in gene expression in HT1080 fibroblasts following treatment with purified recombinant wild-type typhoid toxin (WT-tox) from Salmonella Typhi relative to a mutant H160Q variant lacking DNase activity (HQ-tox), in the presence and absence of serum. The toxin causes DNA damage in both G0/G1 phase and G2/S phase of the cell cycle. Serum-starvation results in host cell cycle arrest in G0/G1 phase, meaning that DNA damage in G2/S is not permissive in the absence of serum. This experiment aimed to uncouple toxin-induced host transcriptomic responses to DNA damage in G0/G1 and G2/S phases of the host cell cycle. Experimental workflow: HT1080 fibroblasts were seeded one day before intoxication into T75 tissue culture flasks for a 30% confluency in DMEM with 1% PenStrep. Cells to be intoxicated in the presence of serum were supplemented with 10% foetal bovine serum (FBS), whereas no FBS was added for serum-starved cells. The next day, cells were incubated for 2h with WT-tox (5 ng/ml) or negative control HQ-tox (5 ng/ml) followed by three washes with PBS and a 48h chase in DMEM supplemented with 1% PenStrep and with or without 10% FBS as appropriate. After 48 h, cells were collected by trypsinisation. RNA was isolated and samples analysed using a human Clariom S assay (ThermoFisher Scientific, 902927). Analysis was performed with Transcriptome Analysis Console 4.0 software (Applied Biosystems, Thermo Fisher Scientific).2025-06-01T00:00:00Z2025-04-17T10:02:22.188Z2025-04-17T10:02:22.188ZE-MTAB-15046EFO_0002768EFO_0002944EFO_0003814EFO_0003813EFO_0005518EFO_0003816EFO_0003815