Project description:The expression of interferon-related genes was more enhanced in irradiated ATM-deficient mouse embryonic fibroblasts (MEFs) than in irradiated ATM wild-type MEFs.
Project description:Head and neck squamous cell carcinoma (HNSCC) is a highly aggressive cancer, with limited therapeutic options and a high mortality rate, primarily due to metastasis and recurrence. Tumor-stroma interactions, and namely cancer-associated fibroblasts (CAFs), are pivotal in shaping HNSCC progression. CAFs remodel the extracellular matrix (ECM) and secrete factors and vesicles that promote tumor growth and metastasis. The interplay between autophagy and endosomal/exosomal pathways has been suggested to regulate cellular secretory functions, but their potential involvement in HNSCC progression remains poorly understood. Since we have recently uncovered Gαq as a key modulator of autophagy, we have investigated the impact of Gαq loss on fibroblast functionality and on its crosstalk with oral HNSCC cells. We report that the absence of Gαq rewires murine embryonic fibroblasts towards CAF-like traits, leading to an increased pro-tumorigenic capacity of co-cultured human oral cancer cells through enhanced collagen I deposition and ECM remodeling. Strikingly, fibroblasts lacking Gαq display a shift the balance of intracellular trafficking, degradative and secretory pathways. Exosomes released from Gαq-deficient fibroblasts show a marked enrichment in tumor-growth factor receptors and can facilitate aberrant tumor growth of HNSCC cells. Gαq-silenced fibroblasts promote the formation of "railroad-tracks" structures around HNSCC cells, enhancing their migratory and invasive capabilities both in vitro and in vivo, and reduced Gαq expression in human HNSCC CAFs correlates with enhanced tumor progression. Overall, our data put forward Gαq as a key regulator of the HNSCC tumor microenvironment by modulating fibroblast plasticity and functionality.
Project description:Exosome-based cell-free therapeutics has received increasing attention in recent decades. Due to the potential demand for therapeutic exosomes, appropriate methods for preservation and storage of exosomes are essential. Current cryopreservation strategies mostly focus on addition of cryoprotectants. However, due to the high concentration of cryoprotectant required, this approach can lead to unfavorable effects. Thus, other storage methods are urgently needed. In this study, we found that Tetraspanin 4 (TSPAN4) and other Tetraspanin family proteins play an essential role in protecting exosomes from cryo-damage. Moreover, we engineered TSPAN4-loaded exosomes which are resistant to cryo-damage. These engineered exosomes show similar properties to wild-type exosomes in protein composition, uptake by recipient cells, and cargo delivery efficiency. We believe our strategy of exosome cryopreservation, without the need for additional agents, will promote the clinical translation of exosomes as therapeutic agents.
Project description:We found that cardiac fibroblasts produce and secrete exosomes. miRNA profiling and TaqMan qRT-PCR experiments identified miR-21 expression to be higher in cardiac fibroblasts compared to those of miR-21*, whereas in exosomes miR-21* expression was higher compared to miR-21. The purpose of the study was to validate these findings by miRNA sequencing in cardiac fibroblasts and fibroblasts-derived exosomes. Neonatal rat cardiac fibroblasts were cultured in DMEM + 1% exosome-depleted FBS for 48h. Conditioned medium was collected and exosomes were purified by several centrifugation and filtration steps, following ultracentrifugation. Afterwards total RNA from cardiac fibroblasts and exosomes was isolated for miRNA sequencing.
Project description:Microarray analysis of differentially expressed genes in wild-type and NFAT5-deficient hematopoietic stem cells (HSC) and multipotent progenitors (MPP)
Project description:RNAseq differential gene expression profiling of olfactory mucosa in young wild type, ACE2 deficient mice. Purpose: Next-generation gene expression profiling has revolutionized analysis of molecular pathways. The goals of this study were to compare NGS-derived olfactory mucosa transcriptome profiles (RNAseq) of wild-type mice (control) and ACE2 knockout mice. Selected genes and pathways will be analyze further by low-throughput techniques such as real-time RT-PCR. Methods: Olfactory mucosa mRNA profiles of 2 months old wild-type (WT) and ACE2 knockout mice were generated by deep sequencing, in triplicate, using Illumina NovaSeq6000 S4 PE150 XP. For inspecting the quality of RNA-Seq data, the 100 most abundant genes are taken from all the samples and heatmaps were generated to observe the relation between samples/conditions. Results: Using our data analysis workflow, we mapped at least 47 million sequence reads per sample to the mouse genome (build mm10). Conlusion: Our study represent the first detailed analysis of differential gene expression by RNAseq technology in murine olfactory mucosa. It is also the first study examining effects of gene knockout for ACE2 on gene expression profile in murine olfactory mucosa. We conclude that these data would help future studies in olfactory mucosa cells aimed to reveal molecular mechanisms associated with aging and biological function of ACE2 genes. RNAseq differential gene expression profiling of olfactory mucosa in wild type and ACE2 deficient mice.
Project description:The expression of interferon-related genes was more enhanced in irradiated ATM-deficient mouse embryonic fibroblasts (MEFs) than in irradiated ATM wild-type MEFs. Nonirradiated-ATM-WT vs Irradiated-ATM-WT vs Nonirradaited-ATM-KO vs IrradiatedATM-KO
Project description:RNAseq differential gene expression profiling of olfactory mucosa in young wild type, aged wild type, APP deficient, APLP2 deficient and PSEN2 deficient mice. Purpose: Next-generation gene expression profiling has revolutionized analysis of molecular pathways. The goals of this study were to compare NGS-derived olfactory mucosa transcriptome profiles (RNAseq) of aged wild-type mice, APP knockout mice, APLP2 knockout mice and PSEN2 knockout mice with young wild-type controls. Selected genes and pathways will be analyze further by low-throughput techniques such as real-time RT-PCR. Methods: Olfactory mucosa mRNA profiles of 2 months old wild-type (WT), 2 years old wild-type, APP knockout, APLP2 knockout and PSEN2 knockout mice were generated by deep sequencing, in triplicate, using Illumina NovaSeq6000 . For inspecting the quality of RNA-Seq data, the 100 most abundant genes are taken from all the samples and heatmaps were generated to observe the relation between samples/conditions. The sequence reads that passed the quality filters were analyzed at the transcript isoform level with TopHat followed by Cufflinks. Results: Using our data analysis workflow, we mapped at least 30 million sequence reads per sample to the mouse genome (build mm10) and identified approximately 25,867 transcripts in the olfactory epithelium of WT and genetically modified mice with TopHat workflow. RNAseq data confirmed stable expression of 20 known housekeeping genes, and 3 of them were validated with qRT-PCR. Approximately 20% of transcripts showed differential expression between WT and aged samples and 0.1-1.0 % showed differential expression between WT and genetically modified lines (fold change >1.5; p value < 0.05). Altered expression of 20 genes for aged samples and 10 genes for PS-deficient samples was confirmed by qRT-PCR, demonstrating the high degree of sensitivity of RNAseq approach. Conlusion: Our study represent the first detailed analysis of differential gene expression by RNAseq technology in murine olfactory mucosa in aged animals. It is also the first study examining effects of gene knockout for APP, APLP2 and PSEN2 on gene expression profile in murine olfactory mucosa. We conclude that these data would help future studies in olfactory mucosa cells aimed to reveal molecular mechanisms associated with aging and biological function of APP, APLP2 and PSEN2 genes. RNAseq differential gene expression profiling of olfactory mucosa in young wild type, aged wild type, APP deficient, APLP2 deficient and PSEN2 deficient mice. Purpose: Next-generation gene expression profiling has revolutionized analysis of molecular pathways. The goals of this study were to compare NGS-derived olfactory mucosa transcriptome profiles (RNAseq) of aged wild-type mice, APP knockout mice, APLP2 knockout mice and PSEN2 knockout mice with young wild-type controls. Selected genes and pathways will be analyze further by low-throughput techniques such as real-time RT-PCR. Methods: Olfactory mucosa mRNA profiles of 2 months old wild-type (WT), 2 years old wild-type, APP knockout, APLP2 knockout and PSEN2 knockout mice were generated by deep sequencing, in triplicate, using Illumina NovaSeq6000 . For inspecting the quality of RNA-Seq data, the 100 most abundant genes are taken from all the samples and heatmaps were generated to observe the relation between samples/conditions. The sequence reads that passed the quality filters were analyzed at the transcript isoform level with TopHat followed by Cufflinks. Results: Using our data analysis workflow, we mapped at least 30 million sequence reads per sample to the mouse genome (build mm10) and identified approximately 24,000 transcripts in the olfactory epithelium of WT and genetically modified mice with TopHat workflow. RNAseq data confirmed stable expression of 20 known housekeeping genes, and 3 of them were validated with qRT-PCR. Approximately 20% of transcripts showed differential expression between WT and aged samples and 0.1-1.0 % showed differential expression between WT and genetically modified lines (fold change >1.5; p value < 0.05). Altered expression of 20 genes for aged samples and 10 genes for PS-deficient samples was confirmed by qRT-PCR, demonstrating the high degree of sensitivity of RNAseq approach. Conlusion: Our study represent the first detailed analysis of differential gene expression by RNAseq technology in murine olfactory mucosa in aged animals. It is also the first study examining effects of gene knockout for APP, APLP2 and PSEN2 on gene expression profile in murine olfactory mucosa. We conclude that these data would help future studies in olfactory mucosa cells aimed to reveal molecular mechanisms associated with aging and biological function of APP, APLP2 and PSEN2 genes. This RNAseq project has been supported by a grant of Polish National Science Centre (UMO-2013/09/NZ3/02359).