Transient genetic modification of plant protoplasts is a straightforward and rapid technique for the analysis of numerous aspects of plant biology. One drawback in the analysis of transformed protoplast suspensions is that they are a heterogeneous mix of cells that have and have not been successfully transfected. To overcome this problem, we have developed a system that employs a fluorescent positive selection marker in combination with flow cytometric analysis as well as fluorescence activated cell sorting (FACS) to isolate responses in the transfected protoplasts exclusively. This recombinase-compatible system enables high-throughput screening of genetic circuitry. Moreover, the use of FACS allows in depth downstream analysis. Lastly, over-expression is an effective means to dissect regulatory networks, especially where redundancy exists. Here, this system has been applied to the study of auxin signaling in order to investigate reporter gene activation and genome-wide transcriptional changes in response to manipulation of the auxin-response network. We have transiently over-expressed dominant negative mutant isoforms of Aux/IAA transcription factors (IAA7mII and IAA19mII; Tiwari et al., 2001) in Arabidopsis Pwer::GFP root protoplasts, making use of a RFP fluorescent positive selection marker and FACS to isolate the dually labeled (IAAnmII expressing and Pwer::GFP-positive) cells. We have compared the transcriptional differences between an empty vector control, IAA7mII and IAA19mII protoplasts that had either been treated with 5microM IAA or mock-treated for 3 hours.
Positive fluorescent selection permits precise, rapid, and in-depth overexpression analysis in plant protoplasts.
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View SamplesWe performed an analysis of transcriptomic responses to auxin within four distinct tissues of the Arabidopsis thaliana root. This high-resolution dataset shows how different cell types are predisposed to react to auxin with discrete transcriptional responses. The sensitivity provided by the analysis lies in the ability to detect cell-type specific responses diluted in organ-level analyses. This dataset provides a novel resource to examine how auxin, a widespread signal in plant development, influences differentiation and patterning in the plant through tissue-specific transcriptional regulation.
A map of cell type-specific auxin responses.
Specimen part, Treatment
View SamplesTo identify potential transient interactions between a TF and its targets, we developed an approach that can identify primary targets based either on TF-induced regulation or TF-binding, assayed in the same samples. Our studies focused on the TF bZIP1 (BASIC LEUCINE ZIPPER 1), a central integrator of cellular and metabolic signaling.
Hit-and-run transcriptional control by bZIP1 mediates rapid nutrient signaling in Arabidopsis.
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View SamplesDevelopmental transitions can be described in terms of morphology and individual genes expression patterns, but also in terms of global transcriptional and epigenetic changes. Most of the large-scale studies of such transitions, however, have only been possible in synchronized cell culture systems. Here we generate a cell type specific transcriptome of an adult stem-cell lineage in the Arabidopsis leaf using RNA sequencing and microarrays. RNA profiles of stomatal entry, commitment, and differentiating cells, as well as of mature stomata and the entire aerial epidermis give a comprehensive view of the developmental progression.
Transcriptome dynamics of the stomatal lineage: birth, amplification, and termination of a self-renewing population.
Specimen part
View SamplesDevelopmental transitions can be described in terms of morphology and individual genes expression patterns, but also in terms of global transcriptional and epigenetic changes. Most of the large-scale studies of such transitions, however, have only been possible in synchronized cell culture systems. Here we generate a cell type specific transcriptome of an adult stem-cell lineage in the Arabidopsis leaf using RNA sequencing and microarrays. RNA profiles of stomatal entry, commitment, and differentiating cells, as well as of mature stomata and the entire aerial epidermis give a comprehensive view of the developmental progression.
Transcriptome dynamics of the stomatal lineage: birth, amplification, and termination of a self-renewing population.
Specimen part
View Samplessorafenib is the treatment of reference for hepatocellular carcinoma (HCC). We applied sorafenib on the human HCC cell line Huh7 and the subclone shRb, carrying a stable knock-down of the expression of the RB1 gene, a key regulator of liver carcinogenesis. Our aim was to better understand the physiologic and metabolic consequences of the exposure of HCC cells to sorafenib.
Metallothionein-1 as a biomarker of altered redox metabolism in hepatocellular carcinoma cells exposed to sorafenib.
Specimen part, Cell line, Treatment
View SamplesThis SuperSeries is composed of the SubSeries listed below.
Global DNA Hypomethylation in Epithelial Ovarian Cancer: Passive Demethylation and Association with Genomic Instability.
Sex, Age, Specimen part, Disease stage
View SamplesComparison of DNA methylome, mRNA transcriptome, and copy number variation in tumors with global loss of DNA methylation to tumors with normal global methylation.
Global DNA Hypomethylation in Epithelial Ovarian Cancer: Passive Demethylation and Association with Genomic Instability.
Sex, Age, Specimen part, Disease stage
View Samplesp53 is a frequent target for mutation in human tumors and previous studies have revealed that these missense mutant proteins can actively contribute to tumorigenesis. To elucidate how mutant p53 might contribute to mammary carcinogenesis we employed a three-dimensional (3D) culture model. In 3D culture non-malignant breast epithelial cells form structures reminiscent of acinar structures found in vivo, whereas breast cancer cells form highly disorganized and in some cases invasive structures. We found that mutant p53 depletion is sufficient to phenotypically revert breast cancer cells to a more acinar-like morphology. Genome-wide expression analysis identified the sterol biosynthesis, or mevalonate, pathway as significantly upregulated by a tumor-derived mutant p53. Using statins and sterol biosynthesis intermediates, we demonstrate that this pathway is both necessary and sufficient for the phenotypic effects of mutant p53 on breast tissue architecture. Mutant p53 associates with the sterol gene promoters at least partly via the SREBP transcription factors. Finally, p53 mutation correlates with higher levels of sterol biosynthesis genes in human breast tumors. This activity of mutant p53 not only contributes insight into breast carcinogenesis, but also implicates the mevalonate pathway as a new therapeutic target for tumors bearing such mutations in p53.
Mutant p53 disrupts mammary tissue architecture via the mevalonate pathway.
Specimen part, Cell line
View SamplesThis SuperSeries is composed of the SubSeries listed below.
Identification of tissue-specific transcriptional markers of caloric restriction in the mouse and their use to evaluate caloric restriction mimetics.
Sex, Specimen part
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