Project description:Membrane proteins are crucial targets for cancer biomarker discovery and drug development. However, in addition to the inherent challenges of hydrophobicity and low abundance, complete membrane proteome coverage of clinical specimen is usually hindered by the requirement of large amount of starting materials. Towards comprehensive membrane proteomic profiling for small amounts of samples (< 10 µg), we developed high-pH reverse phase (Hp-RP) combined with stop-and-go extraction tip (StageTip) technique, as a fast (~ 15 mins.), sensitive, reproducible, high-resolution and multiplexed fractionation method suitable for accurate quantification of the membrane proteome. This approach provided almost 2-fold enhanced detection of peptides within transmembrane helix domains compared with strong anion exchange (SAX) and strong cation exchange (SCX) StageTip techniques. More than 5000 proteins (63% membrane proteins) can be identified in only 10 µg of membrane protein digests, showing the superior sensitivity of the Hp-RP StageTip approach. The Hp-RP StageTip method enabled in-depth membrane proteome profiling of 11 lung cancer cell lines harboring different EGFR mutation status, which resulted in the identification of 3983 annotated membrane proteins. This provides the largest collection of reference peptide spectral data for lung cancer membrane sub-proteome. Finally, relative quantification of membrane proteins between Gefitinib-resistant and -sensitive lung cancer cell lines revealed several upregulated membrane proteins with key roles in lung cancer progression.
Project description:Treatment with the histone deacetylase inhibitor trichostatin a (TSA) changes the radial positioning of the CFTR gene in HeLa S3 cells. The gene relocates from the nuclear periphery to the nuclear interior. In Calu-3 cells the gene is located in the nuclear interior. To identifiy potential regulatory elements for the positioning of CFTR, the histone h3 and h4 acetylation patterns of untreated and TSA-treated HeLa S3 and untreated Calu-3 cells were determined by ChIP-chip. A CTCF site close to the CFTR promoter displayed consistent histone H3 hyperacetylation in TSA treated HeLa S3 cells and Calu-3 cells.
Project description:We report the results of chromatin immunoprecipitation following by high-thoughput tag sequencing (ChIP-Seq) using the GA II platform from Illumina for the human transcription factor STAT1 in HeLa S3 cells. The STAT1 ChIP was performed using HeLa S3 cells that are stimulated using gamma-interferon. We have also generated a seqenced input DNA dataset for gamma-interferon stimulated HeLa S3 cells. Raw data for this study is available for download from the Short Read Archive database at: http://www.ncbi.nlm.nih.gov/Traces/sra/sra.cgi?study=SRP000703. For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf
Project description:The goal of this study was to define how loss of full-length DEK reshapes global transcriptional programs in HeLa S3 cells by comparing gene-expression profiles of wild-type (WT) HeLa S3 and the 1E7 DEK-knockout cells generated by TALEN-mediated genomic disruption. The 1E7 clone lacks detectable full-length DEK, while retaining a low-abundance truncated DEK species, consistent with an alternative transcriptional start site/allelic expression.