Project description:A doxycyline-inducible INS-1 insulinoma cell line expressing proinsulin (C96Y)-GFP was engineered. Addition of doxycyline causes the production of the proinsulin (C96Y)-GFP, which is retained in the endoplasmic reticulum. This study analyzes the gene expression changes that occur after doxycyline-induced expression of proinsulin (C96Y)-GFP for 24h, 48h and 5 days. Expression changes were compared between control un-induced cells and cells treated with doxycyline. Three replicates (experiments) were performed for each time point.
Project description:A doxycyline-inducible INS-1 insulinoma cell line expressing proinsulin (C96Y)-GFP was engineered. Addition of doxycyline causes the production of the proinsulin (C96Y)-GFP, which is retained in the endoplasmic reticulum. This study analyzes the gene expression changes that occur after doxycyline-induced expression of proinsulin (C96Y)-GFP for 24h, 48h and 5 days. Expression changes were compared between control un-induced cells and cells treated with doxycyline. Three replicates (experiments) were performed for each time point. Stable mutant insulin-expressing insulinoma cells were treated or not with doxycyline for 24h, 48h or 5 days and RNA was extracted for expression analysis.
Project description:The Akita mutation (C96Y) in the insulin gene results in early onset diabetes in both humans and mice. Expression of the mutant proinsulin (C96Y) causes endoplasmic reticulum (ER) stress in pancreatic ?-cells and consequently the cell activates the unfolded protein response (UPR). Since the proinsulin is terminally misfolded however, the ER stress is irremediable and chronic activation of the UPR eventually activates apoptosis in the cell population. We used microarray gene expression arrays to analyze the IRE1-dependent activation of genes in response to misfolded proinsulin expression in an inducible mutant proinsulin (C96Y) insulinoma cell line by inhibiting the IRE1 endoribonucleas activity with a specific inhibitor, 4u8c. Insulinoma cells with doxycycline inducible C96Y-proinsulin expression were either untreated, treated with doxycycline alone or treated with dox and 4u8c. This was done with two biological replicates.
Project description:The Akita mutation (C96Y) in the insulin gene results in early onset diabetes in both humans and mice. Expression of the mutant proinsulin (C96Y) causes endoplasmic reticulum (ER) stress in pancreatic -cells and consequently the cell activates the unfolded protein response (UPR). Since the proinsulin is terminally misfolded however, the ER stress is irremediable and chronic activation of the UPR eventually activates apoptosis in the cell population. We used microarray gene expression arrays to analyze the IRE1-dependent activation of genes in response to misfolded proinsulin expression in an inducible mutant proinsulin (C96Y) insulinoma cell line by inhibiting the IRE1 endoribonucleas activity with a specific inhibitor, 4u8c.
Project description:This SuperSeries is composed of the following subset Series:; GSE1589: Targets of HNF1b, HNF4a2 and HNF6 in INS-1 cells; GSE1590: Targets of HNF1b mutants in INS-1; GSE1591: INS-1 cell lines (FLP-In T-REx) Experiment Overall Design: Refer to individual Series
Project description:This study aims at elucidating how Coxsackie B virus infection perturbs the host's miRNA regulatory pathways that may lead to different pathological events using the miRNA microarray approach. The rat pancreatic cell line - INS-1E, was infected with various preparations of Coxsackie B4 viruses was analysed for miRNA expression profiles subsequently. The miRNA expression profiles were measured at 48, and 72 hours post infection, respectively.
Project description:Background Insulinoma is the most common pancreatic neuroendocrine tumour in dogs and humans. The understanding of driving factors and critical survival genes in insulinomas is limited and overall survival is poor for canine and human malignant insulinoma. This study aimed to use single-cell RNA-sequencing to conduct a multispecies analysis of insulinoma cell lines to understand their single-cell transcriptomic landscape. Secondly, the impact of freeze-thawing on the pancreatic beta single-cell transcriptome was investigated, to determine whether cryoarchiving of primary insulinoma samples may be feasible in future studies. Methods Single-cell transcriptomic analysis was performed using fresh and cryopreserved multispecies insulinoma cell lines (canINS, CM, INS-1 and MIN6). R and Seurat were used to perform cell clustering and specific cluster marker genes were identified by the FindMarkers function. Metascape was used to identify statistically enriched pathways for specific cell clusters. Differentially expressed genes between fresh and cryopreserved single-cell transcriptome profiles, were defined as genes with a log2 fold change >0.25 and a Bonferroni-adjusted P<0.05, based on the Wilcoxon rank sum test. Results Based on the specific cell line single-cell transcriptome profiles, five or six cell clusters were constructed per cell line. All cell lines expressed neuroendocrine markers and additionally INS-1 and MIN6 displayed a gene signature indicative of mature/functional pancreatic islet/beta-cells. DEPTOR, BICC1, GHR, CCNB2, CENPA, LMO4, VANGL1, and L1CAM were identified as cross-species conserved insulinoma cluster marker genes. Little effect was found of cryopreservation and thawing on overall gene expression at the single-cell level in insulinoma cell lines: only 6 and 29 genes had a log2 fold change > 1 in cryopreserved versus fresh canINS and CM, respectively. Conclusions canINS, CM, INS-1 and MIN6 are all principally relevant as insulinoma models and the demonstrated differences in their single-cell transcriptomic profiles could aid researchers in selecting the appropriate cell lines for specific study objectives. Cross-species conserved insulinoma cluster marker genes were identified that harbour oncogenes and their involvement in insulinoma tumourigenesis should be investigated in future studies. The good comparability between cryopreserved and fresh insulinoma cells allows for inclusion of cryopreserved insulinoma patient samples in future studies, which allows for reduced assay-based variability.