Dataset Information

Analysis of the cellular effect of 1α,25-dihydroxyvitamin D3 on lung NCI-H441 cells

ABSTRACT: Low serum levels or deficiency of 1α,25-dihydroxyvitamin D3 (VD3) are associated with a higher mortality in trauma patients with sepsis or acute respiratory distress syndrome, although the molecular mechanisms behind this observation are not yet understood. VD3 is known to stimulate lung maturity, alveolar type II cell differentiation and pulmonary surfactant synthesis. This study aims to expand the knowledge by quantitative characterization of NCI-H441 cells upon VD3 treatment at the proteome level.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Lung, Epithelial Cell

DISEASE(S): Severe Acute Respiratory Syndrome

SUBMITTER: Andreas Tholey

LAB HEAD: Andreas Tholey

PROVIDER: PXD040622 | Pride | 2023-05-10

REPOSITORIES: Pride

ACCESS DATA

Publications

Alveolar-Capillary Barrier Protection In Vitro: Lung Cell Type-Specific Effects and Molecular Mechanisms Induced by 1α, 25-Dihydroxyvitamin D3.

Xiong Junyu J Kaleja Patrick P Ückert Larissa L Nezaratizadeh Niloufar N Krantz Stefanie S Krause Martin Friedrich MF Fitschen-Oestern Stefanie S Seekamp Andreas A Cassidy Liam L Tholey Andreas A Fuchs Sabine S

International journal of molecular sciences 20230414 8

Low serum levels of 1α, 25-dihydroxyvitamin D3 (VD3) are associated with a higher mortality in trauma patients with sepsis or ARDS. However, the molecular mechanisms behind this observation are not yet understood. VD<sub>3</sub> is known to stimulate lung maturity, alveolar type II cell differentiation, or pulmonary surfactant synthesis and guides epithelial defense during infection. In this study, we investigated the impact of VD<sub>3</sub> on the alveolar-capillary barrier in a co-culture mod ...[more]

PMID: 37108455

Similar Datasets

Project description:Polycomb repressive complex-2 (PRC2) is a histone methyltransferase required for epigenetic silencing during development and cancer. Long non-coding RNAs (lncRNAs) recruit PRC2 to chromatin, but the general role of RNA in maintaining repressed chromatin is unknown. ChIP-seq, combined with RNA-seq, indicating that PRC2 is also associated with active genes, but most of these are not regulated by PRC2. These results were complemented by in vitro binding assays measuring the binding constant of human PRC2 to various RNAs and find comparable affinity for human lncRNAs targeted by PRC2 and irrelevant transcripts from ciliates and bacteria. PRC2 binding is size-dependent, with lower affinity for shorter RNAs. These findings support a model in which promiscuous binding of PRC2 to RNA transcripts allows it to scan for target genes that have escaped repression, leading to maintenance of the repressed state. Such RNAs may also provide a decoy for PRC2. Cell culture: HEK293T/17 cells were cultured in DMEM with 10% FBS for no longer than 15 passages. ON-TARGETplus SMARTpool for human SUZ12 (Thermo Scientific, Dharmacon cat # L-006957-00-0005) was used to knockdown SUZ12 (siSUZ12) and ON-TARGETplus Non-targeting Pool (Dharmacon cat # D-001810-10-05) was used as a negative control (siCtrl). A total of 25 nM siRNA was transfected in 6-well dishes using LipofectamineM-bM-^DM-" RNAiMAX Reagent (Life Technologies, Invitrogen) following the manufacturerM-bM-^@M-^Ys recommendations. RNA-seq: Polyadenylated RNA was purified from 4 ug of RNA. cDNA libraries were prepared and double-stranded cDNA was fragmented using DNase I according to Illumina specifications, prior to adaptor ligation. Sequencing libraries were amplified and sequenced using an Illumina HiSeq 2000 sequencer. ChIP-seq: 30 million cells at 80% to 90% confluent culture were crosslinked in 1% v/v formaldehyde for 10 min, quenched in 150 mM glycine, washed with cold 1xPBS and harvested by scraping. Cells were lysed in Lysis Buffer (1% w/v SDS, 10 mM EDTA, 50 mM Tris-HCl pH 8.1) with 1x CompleteM-BM-. protease inhibitors (Roche). Cells were sonicated for 10 to 15 min using a BioruptorM-bM-^DM-" UCD-200 (Diagenode) with 30 sec pulses at maximum power. Lysates were diluted to 10 ml in IP Buffer (0.01% w/v SDS, 1.1% v/v Triton-X, 1.2 mM EDTA, 16.7 mM Tris-HCL pH 8.0, 167 mM NaCl) and 10 to 20 ng of antibodies were added and incubated overnight at 4 M-bM-^AM-0C with rotation. Antibodies were immunoprecipitated with 60 M-BM-5l protein G Plus/protein A Agarose Suspension (Calbiochem cat # IP05) and washed sequentially with 1 ml Low Salt Wash Buffer (0.1% w/v SDS, 1% v/v Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0, 150 mM NaCl), 1 ml High Salt Wash Buffer (0.1% w/v SDS, 1% v/v Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0, 500 mM NaCl), 1 ml LiCl Wash Buffer (0.25 M LiCl, 1% v/v Nonidet P-40, 1% w/v deoxycholate, 1 mM EDTA, 10 mM Tris-HCl pH 8.0) and 1 ml TE buffer (Qiagen). DNA was eluted with 0.4 ml of 0.1 M NaHCO3 and 1% w/v SDS. Eluent was transferred into a new tube and NaCl was added to a final concentration of 200 mM. Crosslinks were reversed by incubation at 65 M-bM-^AM-0C for 2 hours. Next, proteins and RNA were digested by adding 33 M-BM-5l of Digestion Reagent (0.6 M Tris-HCl pH 6.5, 152 mM EDTA, 61 ng/ml RNase A (Invitrogen cat # AM2274) and 0.61 mg/ml Proteinase K (NEB cat # P8102)) following by 1 hour incubation at 37 M-bM-^AM-0C. DNA was extracted by phenol:chloroform and ethanol precipitated. DNA was resuspended in Milli-Q pure water and concentration was measured using QubitM-bM-^DM-" (Invitrogen). At least 10 ng of recovered DNA was used to synthesize sequencing libraries using the ChIP-seq Sample Preparation kit (Illumina). Between 6 and 10 pmoles were used for sequencing on the HiSeq2000 sequencer.

Dataset Information

Analysis of the cellular effect of 1α,25-dihydroxyvitamin D3 on lung NCI-H441 cells

Publications

Alveolar-Capillary Barrier Protection In Vitro: Lung Cell Type-Specific Effects and Molecular Mechanisms Induced by 1α, 25-Dihydroxyvitamin D3.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure