Project description:We have investigated the functional effects of DKC1 downregulation in cSCC cells, and unveiled new roles for DKC1 in the metastatic process: metabolism and invasiveness. Transcriptional, proteomic and metabolomic analyses showed that reduced DKC1 levels in cSCC cells resulted in both an activation of the mevalonate pathway and the acquisition of an invasive phenotype.
Project description:The urea channel Slc14a2 (or UT-A1) mediates vasopressin-regulated urea transport across the inner medullary collecting duct (IMCD). Previously, UT-A1 was found to present in a high molecular weight complex, suggesting UT-A1 is involved in certain protein-protein interactions. The present study sought to identify the proteins that interact with UT-A1 in this complex for a better understanding of how UT-A1 is regulated. Rat IMCD suspensions were treated with or without V2 receptor agonist, dDAVP, followed by in-cell crosslinking using BSOCOES and detergent solubilization. Immunoprecipitation using Dynabeads coated with UT-A1 specific antibody successfully pulled down the UT-A1 proteins. In-gel digestion protocol was carried out to prepare samples for liquid chromatographic mass spectrometry analysis of tryptic peptides using a Velos-Orbitrap mass spectrometer. The peptides passing stringent spectral quality thresholds were quantified (label-free) to identify those with (UTA-1 antibody/preimmune IgG) >4. A total of 128 UT-A1 interacting proteins were identified. Gene Ontology analysis maps the distribution of these proteins throughout major cell compartments: endoplasmic reticulum, Golgi, endosomes, cytosol and plasma membrane. Among them are four protein kinases (Cdc42bpb, Phkb, Camk2d, Mtor) that play roles in vasopressin-regulated phosphorylation of UT-A1. Non-label quantification was also performed to determine the stoichiometry of UT-A3 with UT-A1, the result does not support an oligomeric complex formation of UT-A1/A3. In conclusion, we have provided a refined list of UT-A1 binding proteins which can be useful for further analysis of the vasopressin signaling pathway in regulation of UT-A1 in IMCD.
Project description:In this study, we comparatively analyzed the members of the miR-449 family (miR-449a, miR-449b, and miR-449c) with regards to their target genes and functional effects in hepatocellular carcinoma (HCC). Microarray analysis after transient transfection of miR-449a, miR-449b, and/or miR-449c in the HCC cell line HLE identified putative target genes of miR-449a, miR-449b, and miR-449c.
Project description:In this study, we comparatively analyzed the members of the miR-449 family (miR-449a, miR-449b, and miR-449c) with regards to their target genes and functional effects in hepatocellular carcinoma (HCC). Microarray analysis after transient transfection of miR-449a, miR-449b, and/or miR-449c in the HCC cell line HLE identified putative target genes of miR-449a, miR-449b, and miR-449c. For transient overexpression of miRNAs, the HCC cell line HLE was transfected with 50 nM Allstars Negative Control or miScript miRNA mimics. MiRNA mimics for miR-449a, miR-449b, and miR-449c were either single transfected or cotransfected in equimolar amounts (16.7 nM each). Global mRNA expression profiling was performed utilizing pooled RNA of three biological replicates per microarray and two microarrays per condition.
Project description:We model liver phenotypes associated with telomere dysfunction using DC patient-derived iPS cells and isogenic controls with CRISPR/Cas9-mediated homology-directed repair correction of the disease-causing DKC1 mutation. Differentiation of these cells into hepatocyte-like cells or hepatic stellate cells indicates that the parenchymal hepatocytes are primarily affected by telomere dysfunction. We develop an admixed hepatostellate organoid culture model which further reveals that mutant hepatocytes exert dominant effects on hepatic stellate cells regardless of stellate cell genotype. Hepatostellate organoids containing DKC1-mutant hepatocytes exhibit hyperplasia in both the hepatocyte compartment and, remarkably, in stellate cells. Moreover, mutant hepatocytes can induce hallmarks of stellate cell activation independently of stellate cell genotype. Interestingly, mutant hepatostellate organoids also contain off-target PLVAP+ endothelial cells reminiscent of scar-associated endothelium observed in non-DC cirrhosis patients.
Project description:Response of HEK293-cells after transfection with EWS-FLI1. HEK293 cells were transfected with the expression vector pIRES2-EGFP containing type I EWS-FLI1 or empty control vector. For transient transfection standard DEAE dextran method was used and RNA was isolated 48h post transfection. For stable transfection cells were transfected using FuGENE 6 (Roche, Mannheim, Germany) and cells were selected with 400 ug/mL G418. DNA-microarray analysis was performed using Affymetrix HG-U133A microarrays.(see Staege et al. Cancer Res. 2004) Keywords = HEK293 Keywords = EWS-FLI1 Keywords = Ewing family tumors Keywords: other
Project description:Patient-derived glioma stem-like cell (GSC-11), a kind gift of Dr. Lang at UT MD Anderson Cancer Center, was subjected to a transient transfection to down-regulate SOX2 expression. Specific human SOX2 siRNA and a non-targeting control siRNA (si-Scramble) were used in four independent experiments. The cells were then cultured for 72 h after transfection and subjected to the miRNA array analysis.