Project description:The intermediate filament protein Nestin serves as a biomarker for stem cells and has been used to identify subsets of cancer stem-like cells. However, the mechanistic contributions of Nestin to cancer pathogenesis are not understood. Here we report that Nestin binds the hedgehog pathway transcription factor Gli3 to mediate the development of medulloblastomas of the hedgehog subtype. In a mouse model system, Nestin levels increased progressively during medulloblastoma formation resulting in enhanced tumor growth. Conversely, loss of Nestin dramatically inhibited proliferation and promoted differentiation. Mechanistic investigations revealed that the tumor-promoting effects of Nestin were mediated by binding to Gli3, a zinc finger transcription factor that negatively regulates hedgehog signaling. Nestin binding to Gli3 blocked Gli3 phosphorylation and its subsequent proteolytic processing, thereby abrogating its ability to negatively regulate the hedgehog pathway. Our findings show how Nestin drives hedgehog pathway-driven cancers and uncover in Gli3 a therapeutic target to treat these malignancies. Nestin+ and Nestin- GNPs (granule neuron precursors) were purified from Nestin-CFP/Math1-Cre/Ptch1-loxp cerebella at postnatal day 4 by FACs, and total RNA from these two cell populations were extracted, and then labeled and hybridized to Affymetrix Mouse Genome 430 2.0 arrays.
Project description:Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) is an indispensable analytical tool to depict the spatial distribution of analytes such as polar metabolites, lipids, proteins, and drugs in biological specimens. Imaging small polar metabolites and analyzing their in-vivo dynamics with stable isotope labeled (SIL) tracing through various biochemical pathways, including citric acid (TCA) cycle, glycolysis, and amino acid metabolism, has gained substantial interest over the years. However, imaging these polar metabolites across different tissue types is limited due to their lower ionization efficiencies and ion suppression from other abundant biomolecules. These challenges can be further exacerbated with SIL studies, which require the improvement of sample preparation methods. Solvent pretreatments before matrix application on a tissue section have the potential to improve the sensitivity, but often challenges with validation of spatial distribution of metabolites revealed in MALDI-MSI. Here, we present a optimized 'basic hexane' wash method that improved the detection of small molecules with exceptional sensitivity up to several folds for polar and 2H labeled glycolytic metabolites across five different mouse organ tissues (kidney, heart, brain, liver, and brown adipose tissue). Further, we addressed one of the major concerns such as validation of localised polar metabolites within the tissue regions by implementing spatial validation workflow. The workflow depicts the use of region of interest guided laser capture microdissection of tissue regions followed by LC-MS/MS. Indeed, majority of metabolites showed an excellent corroboration with distribution of metabolites in MALDI-MSI and LMD-LC-MS/MS. Besides, we also observed the similar effect on boosting signal intensities of polar metabolites extracted from the microsampled tissues using LMD followed by LC-MS/MS. Overall, we provided an improved MALDI sample pretreatment approach and the spatial validation workflow to understand polar metabolite distribution in mouse organs.
Project description:Transgene loci were transfered in different Arabidopsis thaliana mutant backgrounds by crossing. Small RNA molecules were extracted and sequenced to survey the influence of mutations on transgene small RNA biogenesis.
Project description:The intermediate filament protein Nestin serves as a biomarker for stem cells and has been used to identify subsets of cancer stem-like cells. However, the mechanistic contributions of Nestin to cancer pathogenesis are not understood. Here we report that Nestin binds the hedgehog pathway transcription factor Gli3 to mediate the development of medulloblastomas of the hedgehog subtype. In a mouse model system, Nestin levels increased progressively during medulloblastoma formation resulting in enhanced tumor growth. Conversely, loss of Nestin dramatically inhibited proliferation and promoted differentiation. Mechanistic investigations revealed that the tumor-promoting effects of Nestin were mediated by binding to Gli3, a zinc finger transcription factor that negatively regulates hedgehog signaling. Nestin binding to Gli3 blocked Gli3 phosphorylation and its subsequent proteolytic processing, thereby abrogating its ability to negatively regulate the hedgehog pathway. Our findings show how Nestin drives hedgehog pathway-driven cancers and uncover in Gli3 a therapeutic target to treat these malignancies.
Project description:We report the small RNA transcriptome of testicular extracellular vesicles in mouse testis. We established a testis dissociation protocol to isolate testicular extracellular vesicles. After treatment with proteinase K and RNase A, the RNA inside the extracellular vesicles was extracted and sequenced by small RNA-seq.
Project description:The goal of this study was to perform transcriptomics on vehicle-, UK5099-, Butyrate-, and UK5099+Butyrate-treated mouse prostate organoids. We used FACS to isolate basal cells from C57BL/6 mouse prostates and treated with small molecules for one week before harvesting for RNA-sequencing.