Project description:Patient-derived secondary AML cells were treated with 100 nM of SY-5609 for 24 hours, 100 nM of FHD-286 for 48 hours, or 20 µM of Tasquinimod for 48 hours to determine the global protein expression alterations that correlate with the cell cycle, growth inhibitory and lethal effects of treatment with a CDK7 inhibitor, chromatin remodeling inhibitor, or S100A8/S100A9 inhibitor or in secondary AML cells. Patient-derived de novo AML cells with MLL1 rearrangement and Menin T349M mutation were treated with 100 nM of FHD-286 or 500 nM SNDX-50469 for 48 hours to determine the global protein expression alterations that correlate with the cell cycle, growth inhibitory and/or lethal effects of treatment with a chromatin remodeling inhibitor, FHD-286, or a Menin inhibitor SNDX-50469 in MLL1 rearranged AML cells
Project description:HEL92.1.7 cells in exponential growth phase were treated with 100 nM of SY-5609 for 24 hours to determine the global protein expression alterations that correlate with the cell cycle, growth inhibitory and lethal effects of treatment with a CDK7 inhibitor in secondary AML cells.
Project description:This study examines and compares the protein content in conditioned media collected from neural cell types generated from human pluripotent stem cells. Conditioned media was prepared for 48 hours at a final endpoint of differentiation day 12. Both groups are from parental line WTC11 and cultured as a monolayer on matrigel. Both groups contain a transgene cassette for doxycycline-inducible expression of sox9 and nfia. Doxycycline was only included in the iAstro groups, whereas it was omitted in the neural progenitor cell groups.
Project description:We previously showed that NUDT21-spanning copy-number variations (CNVs) are associated with intellectual disability (ID), and that NUDT21-encoded CFIm25 regulates the protein levels of at least one dose-sensitive, ID-associated protein: MeCP2 (Gennarino et al., 2015). However, the patients’ CNVs also spanned multiple other genes raising the possibility that loss or gain of these other genes caused their symptoms. To determine if reduced NUDT21 function alone is sufficient to cause disease, we generated Nudt21 heterozygous null mice to mimic the human state of reduced expression. We found that although these mice have 50% reduced Nudt21 mRNA, they only have 30% less of its cognate protein, CFIm25. Despite this partial protein-level compensation, the Nudt21+/- mice have learning deficits and cortical hyperexcitability. Further, to determine the molecular mechanism driving neural dysfunction, we partially inhibited NUDT21 in human embryonic stem cell-derived neurons to reduce CFIm25 by 30%. This reduction in CFIm25 was sufficient to induce misregulated alternative polyadenylation (APA) and protein levels in hundreds of genes, dozens of which are associated with intellectual disability and whose dysregulation is likely contributing to disease symptoms. Altogether, these results indicate that disruption of NUDT21-regulated APA events in the brain can cause intellectual disability.
Project description:Inhibition of adipocyte triglyceride lipase (ATGL/PNPLA2) activity in insulin resistance and non-alcoholic fatty liver disease is an attractive therapeutic target. This study evaluated the impact of Atglistatin-mediated ATGL inhibition on non-alcoholic steatohepatitis (NASH) development in diabetic and hyperlipidaemic mice. Streptozotocin-injected male mice were fed an HFD to induce NASH. We analysed liver histology, hepatic lipid content, immunohistochemistry, RNA sequencing, and serum biochemistry. Mechanistically, we treated Caco2 cells, human primary ileum-derived organoids, and HepG2 ATGL KD cells with the novel human ATGL inhibitor NG-497 and WY14643. Atglistatin reduced body and liver weights, total liver lipid content, and improved blood sugar levels consistent with improved liver enzymes, histological liver injury, and NAFLD activity scores. Mechanistically, Atglistatin reduced Cpt1a and Abca1, suggesting impaired Ppara signalling that favours hydrophilic bile acids (BA) synthesis since Cyp7a1, Cyp27a1, and Cyp2c70 were increased while Cyp8b1 was reduced. Accordingly, Intestinal lipid transporters, Abca1 and Cd36, were reduced, consistent with the reduction of liver TAG species, mostly linoleic acids. We used human-specific ATGL inhibitor NG-497 to validate the mouse findings. The NG-497-treated human primary ileum organoids and Caco2 cells showed reduced ATGL, ABCA1, FATP5, CD36, and MTTP. PPARa signalling was impaired in the ileum organoids and HepG2 treated with NG-497 and in HepG2 ATGL KD since PPARa, CPT1a, AOX, ABCA1, and CYP8B1 were reduced, and CYP7A1 increased. Inhibition of ATGL activity reduces the availability of ligands for Ppara activation leading to impaired Ppara signalling, which translate into hydrophilic BA that interferes with dietary lipid absorption, improving metabolic disturbances. The validation with the NG-497 should open a new clinical avenue for NAFLD clinical trial and treatment.
Project description:Our research defines the pivotal roles of the WFDC locus genes in male fertility, particularly in the regulation of sperm development and function. By employing CRISPR/Cas9 mediated gene editing strategy to study this gene family, we provided robust genetic evidence confirming their interconnected functions, which are crucial for sperm differentiation and maturation. Disruptions within these genes can result in infertility, highlighting their regulatory significance. These findings substantiate the potential of targeting EPPIN and related genes for male contraceptive development. Moreover, our study suggests that mutations in the WFDC locus could be instrumental in diagnosing and treating idiopathic male infertility, thereby presenting new therapeutic prospects for reproductive health.
Project description:SUMOylation plays a critical role in regulating protein function. In the ovary, disrupting expression of the E2 SUMO conjugation enzyme in mouse oocytes beginning at the primordial follicle stage results in female sterility and loss of the ovarian reserve in early adulthood. To determine how folliculogenesis is disrupted, we used label-free mass spectrometry to profile day 14 control and Ube2i-Gdf9icre ovaries.
Project description:Proximity labeling discovered a few mitochondrial cristae organizing proteins as NDUFS4 interacting protein. However proximity labeling is unable to differentiate between a direct interaction or a mere close association between NDUFS4 and cristae forming proteins. Additionally, it does not specify if the association of NDUFS4 with these proteins occurs in the context of individual complexes or RSCs. Consequently, to explore whether these cristae forming proteins comigrate and are retained within different complexes in RSCs, we performed complexsome profiling analysis on isolated mitochondria from HG-treated Ndufs4 overexpressed podocytes, with HG-treated cells as control. We separated ETC complexes with BN-PAGE, sliced five distinct bands representing distinct RSCs, labeled 1–5, after Coomassie blue staining, followed by mass spectrometry (LC-MS/MS).
Project description:The cytoplasm is an essential part of the cell involved in protein synthesis, energy generation, nutrient processing, transport, metabolism, and homeostasis, but its structure and function are not entirely understood. Almost five decades ago, dynamic nano-compartments of the cytoplasm were observed by high voltage electron microscopy in every living cell. However, the validity of these constructs, have been questioned and counterclaimed as artifacts of histo-techniques. We developed a new method to separate and characterize two different cytoplasmic compartments that we named cytomatrix and cytosol (or cytomedia). Using two compositional different solvents we were able sequentially extract the cytosol (cyto-solution) first then separate the cytomatrix from the nucleus. Our method allows fractionate the cell into three fractions: the cytosol, cytomatrix, and the nucleus.
Project description:Shotgun proteomic analysis of 3D-grown or on CAM-assay grown lung cancer cells lacking or expressing the major triglyceride lipase adipose triglyceride lipase (ATGL)