This SuperSeries is composed of the SubSeries listed below.
Systematic classification of melanoma cells by phenotype-specific gene expression mapping.
Cell line
View SamplesRecent trials with MAPK inhibitors have shown promising results in many patients with metastatic melanoma; however, nearly all responding patients experience disease relapse. We describe here how melanoma cells respond to MAPK inhibition in a phenotype-specific manner, suggesting that slow cycling invasive phenotype cells provide a treatment-resistant pool from which disease relapse may be derived. The implication is that while MAPK inhibition may successfully treat proliferating cells, another cell population needs to be addressed at the same time.
A proliferative melanoma cell phenotype is responsive to RAF/MEK inhibition independent of BRAF mutation status.
Cell line
View SamplesSOX9 is generally not expressed in melanomas with a high proliferative capacity but is expressed in melanomas with a high invasive capacity. Here we overexpress full length SOX9 in M010817, a melanoma cell culture with high proliferative capacity but low invasive capacity.
Methylation-dependent SOX9 expression mediates invasion in human melanoma cells and is a negative prognostic factor in advanced melanoma.
Specimen part, Cell line
View SamplesHere, we use ribosome-footprint profiing and mRNA-seq to determine the average ribosome density on each gene in S. cerevisiae. We then perform quantitative modeling to identify the molecular determinants of ribosome density. Overall design: Analysis of S. cerevisiae
Poly(A)-tail profiling reveals an embryonic switch in translational control.
Cell line, Subject
View SamplesPolyA Position Profiling (3P-seq) for S. cerevisiae Overall design: Analysis of S. cerevisiae
Poly(A)-tail profiling reveals an embryonic switch in translational control.
Cell line, Subject
View SamplesWe use mRNA-seq in combination with polysome profiling to determine translational status for all mRNAs in Drosophila mature oocytes and activated eggs. Puromycin-treated lysates are used as a negative control in polysome profiling experiments. Additionally, we use ribosome footprinting to globally measure translational efficiency of mRNAs in wild type mature oocytes as well as wild type and png mutant activated eggs. Overall design: Lysates of hand-dissected Drosophila mature oocytes (containing ~540 µg of total RNA) were subjected to separation by velocity sedimentation through sucrose gradients. In this way, free mRNAs (present in RNPs fraction) or those comigrating with ribosomal subunits (40S or 60S+80S fractions) or with varying numbers of bound ribosomes (low polysomes (2-4 ribosomes), medium polysomes (5-9 ribosomes), and heavy polysomes (more than 10 ribosomes) can be separated based on their size and collected as sucrose gradient fractions. To compare quantitatively the levels of every mRNA across the polysome gradient fractions, we added 5ng of S. cerevisiae mRNA as an exogenous spike-in to each of the six fractions of interest: RNPs, 40S, 60S+80S, low polysomes, medium polysomes and heavy polysomes. RNA was extraced from these fractions, follwing proteinase K treatment, by hot acid phenol method. In case of unfractionated lysates, RNA was extracted using TRIzol (Invitrogen) according to manufacturer’s instructions. mRNA-seq samples were prepared from 1 µg of total RNA (in case of sucrose gradient fractions and unfractionated lysates) and subject to Illumina based sequencing. Puromycin-treated lysates of mature oocytes or 0-2h Drosophila activated eggs (containing ~540 µg of total RNA) were also subjected to separation by velocity sedimentation through sucrose gradients. Puromycin causes premature termination of elongating ribosomes and thus it can be used to determine whether the mRNAs co-sedimenting with the polysomal peaks (defined here as =5 ribosomes) were actively engaged in translation. As an independent approach to assess translation and obtain information on the position of ribosomes on mRNAs, we employed ribosome footprinting. In addition to analyzing the same samples, as by polysome profiling, we also analyzed png mutant activated eggs by ribosome footprinting. Ribosome footprint profiling measures the number of ribosome-protected fragments (RPFs) derived from the mRNAs of each gene, resulting in a singular value of translational efficiency (TE) for each gene (TE=RPF/RNA).
Widespread changes in the posttranscriptional landscape at the Drosophila oocyte-to-embryo transition.
Specimen part, Cell line, Subject
View SamplesWe used microarrays to detail the global programme of gene expression in lung SCC cells treated with belinostat, a pan-HDAC inhibitor. The primary focus of this work is to investigate the efficacy of belinostat on lung SCC cells. Our phosphoproteomic profiling analyses revealed the downregulation of MAPK signaling pathway upon drug treatment, together with the induction of apoptosis. While HDAC inhibition generally affects transcription, the mechanism of SOS/MAPK downregulation was therefore proposed to be affected at the transcriptomic level. However, genes related to MAPK pathway were not significantly regulated upon belinostat treatment, whereas ubiquitin-proteasome gene signature was affected. This supports an indirect mechanism of epigenetic regulation on MAPK signaling that should be explored further.
Belinostat exerts antitumor cytotoxicity through the ubiquitin-proteasome pathway in lung squamous cell carcinoma.
Cell line, Treatment
View SamplesThis SuperSeries is composed of the SubSeries listed below.
mRNA destabilization is the dominant effect of mammalian microRNAs by the time substantial repression ensues.
Sex, Age, Specimen part, Cell line, Treatment, Time
View SamplesMicroRNAs (miRNAs) regulate target mRNAs through a combination of translational repression and mRNA destabilization, with mRNA destabilization dominating at steady state in the few contexts examined globally. Here, we extend the global steady-state measurements to many additional mammalian contexts and find that regardless of the miRNA, cell type, growth condition or translational state, mRNA destabilization explains most (70% to >90%) miRNA-mediated repression. We also determine the relative dynamics of translational repression and mRNA destabilization for endogenous mRNAs as a miRNA is induced. Although translational repression occurs rapidly, its effect on gene expression is relatively weak, such that by the time consequential repression ensues, the effect of mRNA destabilization dominates. These results add to the fundamental understanding of miRNAs, imply that consequential miRNA-mediated repression is largely irreversible and simplify future studies, dramatically extending the known contexts and time points for which monitoring mRNA changes captures most of the direct miRNA effects.
mRNA destabilization is the dominant effect of mammalian microRNAs by the time substantial repression ensues.
Sex, Age, Specimen part
View SamplesMammalian gonadal sex determination is dependent on proper expression of sex determining genes in fetal gonadal somatic support cells (i.e., pre-granulosa and pre-Sertoli cells in XX and XY gonads, resp.). We used a unique transgenic mouse strain combined with microarray profiling to identify all the differentially expressed transcripts in XX and XY isolated somatic support cells during critical stages of gonadal development and differentiation.
New candidate genes identified for controlling mouse gonadal sex determination and the early stages of granulosa and Sertoli cell differentiation.
Sex, Specimen part
View Samples