Project description:Human hepatocytes, HepaRG cells and HCC HepG2 cells were treated with 50nM of Microcystin-LR and cylindrospermopsin (CYN) to observe changes in gene expression.
Project description:We analyzed the effect of knockdown of miR-214 compared to wild type on the expression of genes and pathways involved in prostate cancer cells.
Project description:We chronically exposed HepaRG (differentiated hepatic cells that retain primary human hepatocytes characteristics) and HepaG2 (liver hepatocarcinoma cells) liver cells to physiologically relevant concentrations of Cadmiun and PFOAs chemicals (10nM) to observed how these affect global gene expression in these cellular models.
Project description:We investigated how selective mutation in STK11 or KEAP1 alters the transcriptional profile of cancer cells, and how the transcriptional profile of co-mutant cells might uniquely affect cancer-related pathways including control of proliferative potential, metabolic homeostasis, and cell death. We performed bulk RNAseq on none, single or double mutation of these genes in single cell knockout clones (n=3 per group) from both H358 and H292 cell lines.
Project description:We found that the fungus, Debaryomyces hansenii (D. hansenii), is enriched in inflamed intestinal tissue from patients of Crohn's disease and its administration to mice impairs colonic wound healing in multiple models of colonic injury and repair. To understand the mechanism, we isolated bone marrow derived macrophages from mice and stimulated them in vitro with a pure isolate of D. hansenii. Total RNA was isolated at multiple time points and assayed for transcriptomic analysis.
Project description:We generated C57BL/6 mice lacking Bmp10 and/or Bmp9 utilizing the Cre-loxP system. Briefly, Bmp9 constitutive deletion resulted from the replacement of exon 2 by a neomycin resistance cassette. Because Bmp10 deletion leads to early embryonic lethality, we used the tamoxifen-inducible Cre system to generate Bmp10-cKO mice (Rosa26-CreERT2;Bmp10lox/lox) by crossing Rosa26-CreERT2 mice with Bmp10lox/lox mice that possess loxP sites flanking exon 2. To generate double-KO (DKO) mice, we crossed these Rosa26-CreERT2;Bmp10lox/lox mice with Bmp9-KO mice. At the age of 8 weeks, mice were treated with tamoxifen (Sigma) by intraperitoneal injection once a day for 5 days at a dosage of 50 mg/kg. At the age of 5 months, Wild Type and DKO mouse lung tissue was flash frozen in liquid nitrogen and stored at -80°C. RNA extraction, RNA sample quality assessment, RNA library preparation, sequencing and raw data analysis were conducted at GENEWIZ, Inc. (South Plainfield, NJ, USA). Total RNA was extracted from frozen tissue using the Qiagen RNeasy Plus Mini kit. RNA samples were quantified using Qubit 2.0 Fluorometer (Life Technologies, Carlsbad, CA, USA) and RNA integrity was checked with Agilent TapeStation (Agilent Technologies, Palo Alto, CA, USA). rRNA depletion was performed using Ribozero rRNA Removal Kit (Illumina, San Diego, CA, USA). RNA sequencing library preparation used NEBNext Ultra RNA Library Prep Kit for Illumina by following the manufacturer’s recommendations (NEB, Ipswich, MA, USA). Briefly, enriched RNAs were fragmented for 15 minutes at 94 °C. First strand and second strand cDNA were subsequently synthesized. cDNA fragments were end repaired and adenylated at 3’ends, and universal adapter was ligated to cDNA fragments, followed by index addition and library enrichment with limited cycle PCR. Sequencing libraries were validated using the Agilent Tapestation 4200 (Agilent Technologies, Palo Alto, CA, USA), and quantified by using Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA) as well as by quantitative PCR (Applied Biosystems, Carlsbad, CA, USA). The sequencing libraries were clustered on one lane of a flowcell. After clustering, the flowcell was loaded on the Illumina HiSeq 4000 instrument (or equivalent) according to manufacturer’s instructions. The samples were sequenced using a 2x150 Paired End (PE) configuration. Image analysis and base calling were conducted by the HiSeq Control Software (HCS). Raw sequence data (.bcl files) generated from Illumina HiSeq was converted into fastq files and de-multiplexed using Illumina's bcl2fastq 2.17 software. One mis-match was allowed for index sequence identification. After investigating the quality of the raw data, sequence reads were trimmed to remove possible adapter sequences and nucleotides with poor quality using Trimmomatic v.0.36. The trimmed reads were mapped to the the Mus musculus GRCm38 reference genome available on ENSEMBL using the STAR aligner v.2.5.2b. Gene counts were calculated from uniquely mapped reads using feature Counts from the Subread package v.1.5.2. Only unique reads that fell within exon regions were counted. The gene hit counts table was then used for downstream differential expression analysis. A differential gene expression analysis between WT and DKO groups of samples was performed using the R-package DESeq2 (Wald test).
Project description:Genomic alterations that activate Fibroblast Growth Factor Receptor 2 (FGFR2) are common in intrahepatic cholangiocarcinoma (ICC), a deadly bile duct malignancy. FGFR kinase inhibitors (FGFRi) have shown promising efficacy against FGFR2+ ICC in clinical trials, leading to the regulatory approval of the ATP-competitive FGFR inhibitors, pemigatinib and infigratinib (BGJ398), for this subset of patients who failed standard treatment . However, the objective response rate (ORR) for each FGFR inhibitor (FGFRi) studied to date in FGFR2+ ICC is <45% and disease progression invariably arises within ~6-12 months. By employing high-throughput drug screens and signaling studies, we identified signaling feedback via the EGFR pathway as a major mediator of adaptive resistance to FGFR kinase inhibition in a set of patient-derived ICC models. To further gain insights into the synergistic effects of the EGFR/FGFR combination and address the mechanisms underlying the survival pathway reactivation, we performed RNA sequencing in our FGFR-driven ICC models (ICC21 and ICC10-6). For these studies, we treated FGFR2 fusion+ ICC cell lines ICC21/ICC10-6 with four conditions (DMSO/Infigratinib/Afatinib/Combo) for 4 hours followed by RNA sequencing.
Project description:The objective of this study was to decipher the metabolism expressed by Lactobacillus delbrueckii subsp. delbrueckii CIRM-BIA865 during soy juice fermentation using transcriptomics. The whole genome was sequenced, assembled and annotated. CIRM-BIA865 was then used to ferment soy juice to produce a soy-based yogurt. Samples were analysed in kinetics during fermentation, at pH values of 6.5, 6, 5 and 4.6. RNA from CIRM-BIA865 were extracted and sequenced using paired-end Illumina. Reads were mapped using Bowtie2 on previously obtained genome of CIRM-BIA865. No mismatch were allowed. Reads mapped on CDS were counted using htseqcount.List of differentially expressed (DE) genes between two successive sampling times (determined by pH) were generated using DEseq2 with a modified t-test and a p-value adjusted by Bonferoni inferior to 0.05. Fold changes expressed how many times genes were induced along the fermentations.
Project description:The aim of the study was to decipher metabolisms responsible (i) for the peculiar adaptation of L. plantarum during soy juice fermentation and (ii) for the release of aroma compounds, amino and short-chain fatty acid, and metabolites with health-promoting properties in soy yogurt. The strategy was the sequencing and annotation of a strain (L. plantarum CIRM-BIA777, PRJEB77707) able to degrade galacto- oligosaccharides, the sampling of soy yogurt, RNA-seq to identify differentially expressed genes of L. plantarum and corresponding metabolisms throughout the kinetics of fermentation. Acids and volatile compounds were also quantified.
Project description:Autotaxin (ATX), encoded by ENPP2, catalyzes the production of lysophosphatidic acid (LPA), an important regulator within the tumor microenvironment (TME). ATX is a clinical target in pancreatic ductal adenocarcinoma (PDAC), yet the pro-tumorigenic action of the ATX/LPA axis in PDAC remains unclear. PDAC is characterized by a highly fibrotic tumor microenvironment (TME) due to an abundance of cancer associated fibroblasts (CAFs), acting as a barrier to therapy and contributing to the poor survival of PDAC patients. The mechanism by which ATX inhibition influences CAFs and their secretome is still not fully characterized. To identified potential downstream mediators of ATX signaling, we performed RNA-seq of pancreatic CAF-derived cell line 0082T treated with Autotaxin inhibitors, IOA-289 and PF-8380. PF-8380 treatment resulted in a higher number of differentially expressed genes compared to IOA-289 treatment. IOA-289 treatment resulted in only four significantly differentially expressed genes (CTGF, PLIN2, HHIP, and AHNAK). However, only PLIN2, which was upregulated and CTGF, which was downregulated, overlapped between the two ATX inhibitors. As CTGF (connective tissue growth factor) is a pro-fibrotic and pro-tumorigenic factor, it suggests a key role for CAF-derived ATX in promoting autocrine and paracrine pro-tumorigenic signaling in PDAC.