Cytoplasmic Accumulation of Incompletely Glycosylated Sex Hormone-binding Globulin (SHBG) Enhances Androgen Action in Proximal Convoluted Tubule Epithelial Cells
ABSTRACT: Identification of androgen-responsive genes that are influenced by the presence of a functional SHBG versus a steroid-binding-deficient SHBG mutant (SHBG S42L) in the cytoplasm of PCT cells. The hypothesis tested in the study was that SHBG enhances androgen action. Results provide important information that intracellular SHBG enhances androgen-dependent stimulation or repression in PCT cells. RNA was extracted for gene expression profiling from PCT cells expressing wild-type human SHBG or the human SHBG S42L mutant after treatment with testosterone or DHT, and hybridized to Illumina Mouse WG-6 v2.0 expression bead chips. Three replicates each.
Project description:Identification of androgen-responsive genes that are influenced by the presence of a functional SHBG versus a steroid-binding-deficient SHBG mutant (SHBG S42L) in the cytoplasm of PCT cells. The hypothesis tested in the study was that SHBG enhances androgen action. Results provide important information that intracellular SHBG enhances androgen-dependent stimulation or repression in PCT cells. Overall design: RNA was extracted for gene expression profiling from PCT cells expressing wild-type human SHBG or the human SHBG S42L mutant after treatment with testosterone or DHT, and hybridized to Illumina Mouse WG-6 v2.0 expression bead chips. Three replicates each.
Project description:This ArrayExpress record contains meta-data and results of quantitative analysis of cell lines from the NCI-60 panel using pressure cycling technology (PCT) and SWATH-mass spectrometry. Each cell line was analyzed in duplicate. Raw data files are available at the EMBL-EBI protemics data archive (PRIDE) at accession PXD003539 (http://www.ebi.ac.uk/pride/archive/projects/PXD003539). Since the record here does not include the raw data files and hence there is no need to explicitly link individual replicate to a raw file, each sample is only listed once in the ArrayExpress samples table for clarity.
Project description:DDA and SWATH analysis of human kidney tissues. Data set used for PCT-SWATH paper. PCT-SWATH provides the first method to generate a digital map representing the proteome of biopsy level clinical samples from which thousands of proteins can be accurately quantified with a high degree of reproducibility across sample sets.
Project description:Here we investigated the degradation of mRNA and protein in 68 pairs of adjacent prostate tissue samples using RNA-seq and pressure cycling technology (PCT) coupled with SWATH mass spectrometry and developed a score, the Proteome Integrity Number (PIN), to quantify the extent of protein degradation in the samples.
Project description:Understanding the progression of periodontal destruction is at the forefront of periodontal research. Therefore, we aimed to capture the dynamics of gingival tissue proteome during initiation and progression of an experimental periodontitis developed by placing silk ligatures on the second molars of the specific pathogen-free C57BL/6 mice. Pressure cycling technology (PCT), a recently developed platform that uses ultra-high pressure to disrupt tissues, was utilized to achieve an efficient and reproducible protein extraction for quantitative shotgun proteomics. Lysis and protein digestion of gingival tissue was performed using PCT and analysed by Orbitrap Fusion. The proteomics data were quantitatively assessed with the Progenesis QI software.
Project description:Androgens drive the onset and progression of prostate cancer (PCa) by modulating androgen receptor (AR) transcriptional activity. Although several microarray-based studies have identified androgen-regulated genes, here we identify in-parallel global androgen-dependent changes in both gene and alternative mRNA isoform expression by exon-level analyses of the LNCaP transcriptome. While genome-wide gene expression changes correlated well with previously-published studies, we additionally uncovered a subset of 226 novel androgen-regulated genes. Gene expression pathway analysis of this subset revealed gene clusters associated with, and including the tyrosine kinase LYN, as well as components of the mTOR (mammalian target of rapamycin) pathway, which is commonly dysregulated in cancer. We also identified 1279 putative androgen-regulated alternative events, of which 325 (~25%) mapped to known alternative splicing events or alternative first/last exons. We selected 30 androgen-dependent alternative events for RT-PCR validation, including mRNAs derived from genes encoding tumour suppressors and cell cycle regulators. Of seven positively-validating events (~23%), five events involved transcripts derived from known AR gene targets. In particular, we found a novel androgen-dependent mRNA isoform derived from an alternative internal promoter within the TSC2 tumour suppressor gene, which is predicted to encode a protein lacking an interaction domain required for mTOR inhibition. We confirmed that expression of the alternative TSC2 mRNA isoform was directly regulated by androgens. Furthermore, by chromatin immunoprecipitation, we observed recruitment of AR to the alternative promoter region at early timepoints following androgen stimulation, which correlated with expression of alternative transcripts. Together, our data suggest that alternative mRNA isoform expression might mediate the cellular response to androgens, and may have roles in clinical PCa. Total 8 samples were analysed. 4 control (LNCaP cells grown in RPMI-1640 media with charcoal-stripped 10% FBS devoid of steroids) and 4 treatment (LNCaP cells grown in media devoid of steroid, and subsequently treated with 10nM R1881 synthetic androgen analogue for 24 hours).
Project description:We obtained quantitative analysis of the NCI-60 cell panels using pressure cycling technology (PCT) and SWATH-MS. Each cell was analyzed in duplicate. The data were analyzed using a cell line SWATH assay library and OpenSWATH, followed by SWATH-expert refinement.
Project description:LNCaP cells were cultures in steroid depleted medium for 5 days before treatment with synthetic androgen (R1881, 10nM) for 16h. Transcriptomics analysis was performed to compare gene expression changes induced by androgen withdrawal or androgen treatment. Genome-wide transcriptomic analysis of LNCaP cells grown in steroid depleted medium, normal (steroid-containing) medium and R1881 treated cells was performed using the Agilent platform