Unknown,Transcriptomics,Genomics,Proteomics

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Salmonella Typhimurium: Heat-shocked cells vs. control cells and acid-shocked cells vs. control cells

ABSTRACT: We investigated if the transcriptional response of S. Typhimurium to temperature and acid variations was hysteretic and showed memory, i.e., if the transcriptional regulation caused by environmental stimuli remained after the stimuli ceased. The transcriptional activity of non-replicating stationary-phase cells of Salmonella Typhimurium caused by the exposure to 45M-BM-0C and to pH 5 for 30 min was monitored by microarray hybridizations before the application of the stress and at the end of the treatment period, as well as immediately and 30 minutes after conditions were set back to their initial values, 25M-BM-0C and pH 7, respectively. We also investigated if hysteresis was accompanied with higher resistance to inactivation conditions, 50M-BM-0C and/or pH 3, as well as more efficient growth at extreme conditions, 43M-BM-0C and/or pH 4.5. RNA samples for gene expression analysis and culture samples for growth and inactivation experiments were obtained after 30 min with stress (45M-BM-0C or pH 5), immediately after the removal of stress and 30 min after the removal of stress. A control culture at 25M-BM-0C and pH 7 was sampled at the same time intervals. Three independent replicates were carried out.

ORGANISM(S): Salmonella enterica subsp. enterica serovar Typhimurium

SUBMITTER: Carmen Pin

PROVIDER: E-GEOD-37636 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:While the regulation of metabolic enzymes by oncogenic drivers or tumor suppressors has been intensively studied over recent years, our understanding of how metabolic processes directly regulate cell proliferation has remained fragmentary. Here we show how the alteration of metabolism directly affects cell cycle progression in cancer cells. We found that activation of the nuclear receptor peroxisome-proliferation activated receptor gamma (PPARM-NM-3), a transcriptional master regulator of lipid metabolism, inhibits the growth of lung adenocarcinoma cells by triggering a metabolic switch that inhibits pyruvate oxidation and reduces glutathione levels. These PPARM-NM-3-induced metabolic changes result in a marked increase of reactive oxygen species (ROS) levels that lead to rapid hypophosphorylation of retinoblastoma protein (RB) and cell cycle arrest. Both of these changes can be prevented by suppressing pyruvate dehydrogenase kinase 4 (PDK4) or M-NM-2-oxidation of fatty acids. Thus, we provide a mechanism that directly links metabolic changes to inhibition of cancer cell cycle progression by altering ROS levels. We generated PPARG-LAP BAC transgenic NCI-H2347 and NCI-H1993 cell lines using the BAC-transgenesis approach. Cells at 80% confluency (~1-1.5x107) were cross-linked with 1% formaldehyde for 10 minutes at 37M-BM-0C, and quenched with 125 mM glycine at room temperature for 5 minutes. The fixed cells were washed twice with cold PBS, scraped, and transferred into 1 ml PBS containing protease inhibitors (Roche). After centrifugation at 700 g for 4 minutes at 4M-BM-0C, the cell pellets were resuspended in 100 M-NM-<l ChIP lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl [pH 8.1] with protease inhibitors) and sonicated at 4M-BM-0C with a Bioruptor (Diagenode) (30 seconds ON and 30 seconds OFF at highest power for 12 minutes). The sheared chromatin with a fragment length of ~200 M-bM-^@M-^S 600 bp) was centrifuged at 10,000 g for 10 minutes at 4M-BM-0C). 100 M-NM-<l of the supernatant was used for ChIP or as input. A 1:10 dilution of the solubilized chromatin in ChIP dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 167 mM NaCl 16.7 mM Tris-HCl [pH 8.1]) was incubated at 4M-BM-0C overnight with 6 M-NM-<g/ml of a goat anti-GFP (raised against His-tagged full-length eGFP and affinity-purified with GST-tagged full-length eGFP). Immunoprecipitations were carried out by incubating with 40 M-NM-<l pre-cleared Protein G Sepharose beads (Amersham Bioscience) for 1 hour at 4M-BM-0C, followed by five washes for 10 minutes with 1ml of the following buffers: Buffer I: 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl [pH 8.1], 150 mM NaCl; Buffer II: 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl [pH 8.1], 500 mM NaCl; Buffer III: 0.25 M LiCl, 1% NP-40, 1% deoxycholate, 1 mM EDTA, 10 mM Tris-HCl [pH 8.1]; twice with TE buffer [pH 8.0]. Elution from the beads was performed twice with 100 M-NM-<l ChIP elution buffer (1% SDS, 0.1 M NaHCO3) at room temperature (RT) for 15 minutes. Protein-DNA complexes were de-crosslinked by heating at 65M-BM-0C in 192 mM NaCl for 16 hours. DNA fragments were purified using QiaQuick PCR Purification kit (Qiagen) and eluted into 30 M-NM-<l H2O according to the manufacturerM-bM-^@M-^Ys protocol after treatment with RNase A and Proteinase K.

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Salmonella Typhimurium: Heat-shocked cells vs. control cells and acid-shocked cells vs. control cells

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