RNA-Seq Analyses of the apoptosis and necroptosis in MEF
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ABSTRACT: Purpose: we used next generation sequencing to analyze gene expression profiles of MEF treated with PBS (control), TNF-ɑ + Cycloheximide (TC) or TNF-ɑ + Cycloheximide+ z-VAD-fmk (TCZ). The goals of this study are to compare the different gene expression profiles between MEF treated with TC compared to PBS treatment and MEF treated with TCZ compared to PBS treatment. Methods: In MEF, the combination of TNF-α and cycloheximide (TC) induces apoptosis, whereas the addition of z-VAD-fmk (TCZ) to this regimen promotes necroptosis, using High-seq 2000 Illumina sequencing platform. The sequence reads that passed quality filters were analyzed at the transcript isoform level with two methods: Burrows–Wheeler Aligner (BWA) followed by ANOVA (ANOVA) and TopHat followed by Cufflinks. qRT–PCR validation was performed using TaqMan and SYBR Green assays Results: By comparing the RPKM values between the two groups, a total 492 DEGs (342 upregulated and 150 downregulated) were identified in MEF treated with TC compared to PBS treatment, and a total 424 DEGs (242 upregulated and 182 downregulated) were identified in MEF treated with TCZ compared to PBS treatment. Next, we compared all identified DEGs in MEF between TC and TCZ treatment. We found that 252 DEGs overlapped between TC and TCZ treatment, whereas 240 DEGs were only present in TC treatment and 172 DEGs were only present in TCZ treatment. Conclusions: we firstly identified 492 DEGs in apoptotic MEF, and as well as 424 DEGs in necroptotic MEF. And we performed the enriched molecular pathway, molecules, transcription factors and signaling networks by IPA analysis. We revealed that apoptosis and necroptosis may share certain common canonical pathways and transcription factors.
Project description:Pan-caspase inhibitor Z-VAD-fmk acts as an inhibitor of peptide:N-glycanase (NGLY1); an endoglycosidase which cleaves N-linked glycans from glycoproteins exported from the endoplasmic reticulum during ER-associated degradation (ERAD). Pharmacological N-glycanase inhibition by Z-VAD-fmk or siRNA knockdown (KD) induces GFP-LC3 positive puncta in HEK293 cells. Activation of ER stress markers or reactive oxygen species (ROS) induction are not observed. In NGLY1 inhibition or KD, upregulation of autophagosome formation without impairment of autophagic flux are observed. Enrichment and proteomics analysis of autophagosomes after Z-VAD-fmk treatment or NGLY1 KD reveals similar autophagosomal protein content. Autophagy represents a cellular adaptation to NGLY1 inhibition or KD, and ATG13-deficient mouse embryonic fibroblasts (MEFs) show reduced viability under these conditions. In contrast, treatment with pan-caspase inhibitor, Q-VD-OPh does not induce cellular autophagy. Therefore, experiments with Z-VAD-fmk are complicated by the effects of NGLY1 inhibition and Q-VD-OPh represents an alternative caspase inhibitor free from this limitation.
Project description:Objective: The knowledge about functions of caspases, traditionally associated with cell death and inflammation, keeps expanding also regarding cartilage. Active caspases are expressed by chondrocytes within the growth plate and caspase inhibition in limb-derived chondroblasts altered osteogenesis-related genes. Caspase inhibitors were reported to reduce the severity of cartilage lesions in osteoarthritis (OA) and caspase-3 might be a promising biomarker for OA prognosis. Design: To provide an overview about impact of caspase inhibition on expression profile in chondroblasts, the whole transcriptome RNA sequencing was performed. Limb-derived chondroblasts were cultured with two different inhibitors, Z-VAD-FMK (FMK) and Q-VD-OPH (OPH). Results: The analysis revealed a statistically significant increase in the expression of 252 genes in the FMK samples and 163 genes in the OPH samples compared to controls. Conversely, there was a significant decrease in the expression of 290 genes in the FMK group and 188 in the OPH group. Among the top up- and down-regulated genes (more than 10 times changed), almost half of them have been associated with OA. Both inhibitors displayed the highest up-regulation of the inflammatory chemokine Ccl5. In the presence of one or the other inhibitor, the most down-regulated gene was the one for mannose receptors Mrc1. Conclusion: The results of this investigation yielded not only a list of genes associated with OA as being up- or down-regulated, but also a comprehensive expression profile in primary chondroblasts after general caspase inhibition. Additionally, comparison of the inhibitory impact in the case of FMK inhibitor vs. OPH inhibitor was provided.
Project description:Isolated human hepatocytes are used in all facets of liver research, from the clinical management of liver failure to in vitro studies of drug disposition. Cryopreservation provides a reliable source of hepatocytes, but the associated cellular stress causes a highly variable and heterogeneous loss of a differentiated phenotype, manifested by a decreased ability to form cell-matrix and cell-cell interactions. We reasoned that this problem could be mitigated at the post-thawing stage, which would increase the availability of well-functioning human hepatocytes. We applied quantitative global proteomics to analyze the differences between attached and non-attached fractions of cryopreserved human hepatocyte batches. Hepatocytes that were unable to attach to a collagen matrix showed many signs of cellular stress, including a glycolytic phenotype and activation of the heat shock response, with increased apoptosis activation as the ultimate consequence. Further analysis of the activated stress pathways revealed an increase in early apoptosis immediately after thawing, hinting at the possibility of stress reversal. Therefore, we transiently treated the cells with compounds aimed at decreasing cellular stress via different mechanisms. We found that brief exposure to the pan-caspase apoptosis inhibitor Z-VAD-FMK restored the ability to attach to collagen, and promoted a differentiated morphology with increased metabolic function. Further, Z-VAD-FMK treatment did not alter hepatocyte protein expression, suggesting that these ‘rescued’ cells would be suitable for applications where hepatocytes of high quality are required. Conclusion: Cryopreserved human hepatocytes are affected by considerable amounts of cellular stress, often resulting in loss of differentiation with a decreased ability to form cell-matrix and cell-cell interactions. This can be alleviated by brief apoptosis inhibition post-thawing, substantially improving key morphological and functional properties of these cells.
Project description:In this study, we investigated the miRNA and mRNA profiling of the cortex of rat model of vascular dementia (VaD) to analyze the regulatory mechanism in the pathology of VaD involved by miRNAs, transcription factors (TFs), and corresponding target genes. As a validated model of VaD, rats suffering from bilateral common carotid artery occlusion (2VO) were used in the present study and the reliability of this model was examined by the Morris water maze (MWM) test and the Nissl staining. Overall, results showed that rats with 2VO presented declined learning and memory capabilities in the MWM test and neuronal loss in the hippocampus and cortex indicated by Nissl-staining compared to sham rats. DEGs, DEMs, and DETFs were discriminated between rats with 2VO and sham rats in the cortex, illustrated by 13 aberrantly-expressed miRNAs, 805 mRNAs, and 63 TFs. Further network analysis revealed that 7 target genes, 5 miRNAs, and 10 TFs were the key molecule in the miRNA-TF-gene network related to VaD. Gene Ontology (GO) and pathway enrichment analyses of these VaD-related transcripts showed that these differently changed genes mostly got involved in PI3K-Akt signaling pathway, neuroactive ligand-receptor interaction, calcium signaling pathway, and Wnt signaling pathway, along with central locations around cell membrane, exerting function such as growth factor binding, integrin binding, and extracellular matrix structural constituent, with representative biological processes like vasculature development, cell-substrate adhesion, cellular response to growth factor stimulus, and synaptic transmission. In conclusion, these results will help us understand the underlying regulatory mechanisms of miRNA-TF-genes in pathogenesis and provide potential therapeutic targets for the treatment of VaD.
Project description:To get insight of molecular mechanism of GPR39, we investigated transcriptomic response in GPR39 overexpressed HEK293 cells after TC-G-1008 stimulation We identified 814 differentially expressed genes (DEGs) fold change cutoff = 1.5 in the GPR39 overexpressed HEK293 and GPR39 overexpressed HEK293 cells with TC-G-1008 treatment, those including 364 up-regulated and 450 down-regulated genes These findings suggest that MAPK/Erk, PI3K/Akt and Glycerolipid metabolism signaling pathways would be putative signaling pathways dominantly altered by GPR39 activation.
Project description:In chicken DT40 cells, there are six linker histone H1 variants and 12 of coding genes. We have previously reported of 11 out of 12 H1 knock out DT40 cells (Takami et al., Genes to Cell 1997 [PMID:9491804]) but complete H1 null DT40 cells could not established, so far. We identified one of the H1 variant, H1R was involved in genomic instabilities (Hashimoto et al., DNA repair (2007) [17613284]), so we re-introduced floxed H1R-eGFP and mer-cre-mer into 11 out of 12 H1 knock out DT40 cells. Then we targeted last enedogenous H1, we successfully established conditional H1 KO cells (K11). Next we treated with tamoxifen to loop out floxed H1R-eGFP, and cloning H1 completely null cells (K11-5, and K11-7). We analysis those gene expression pattern in wild-type, K11, and K11-5 cells Experiment Overall Design: Apoptosis is induced in H1 null cells, so we inhibit apoptosis with pan-caspase inhibitor, Z-VAD-FMK and extract RNAs.
Project description:Alzheimer’s disease (AD) and vascular dementia (VaD) are the two most common forms of dementia, neither of which can be effectively treated. There is growing evidence on vascular contributions to cognitive impairment and dementia such as AD, but their pathogenic molecular links are not defined yet. Notably, neurofibrillary tangles made of hyperphosphorylated tau (P-tau) are a hallmark lesion of AD, but are not found in VaD. Although brain ischemia induces some tau changes and tau knockout reduces stroke-induced acute brain damage, little is known about the role of tau in mediating progression from vascular insufficiency to later development of VaD. Cis P-tau is a pre-tangle pathology in AD and an early driver of neurodegeneration resulting from brain injury, but its role in AD treatment or in VaD is unknown. Here we identify cis P-tau as an antibody-neutralizable major common early driver of AD and VaD. We show that cis P-tau elimination using cis antibody not only prevents, but also treats AD-like neurodegeneration and memory loss in a hTau mouse model of AD. Purified cis P-tau causes and spreads neurodegeneration with behavioral changes when injected into wild-type mouse brains, but is prevented by cis antibody. Surprisingly, we also find robust cis P-tau with no evidence of tau tangles in human VaD brains and a mouse model of chronic cerebral hypoperfusion that mimics key aspects of clinical VaD. Cis mAb treatment of hypoperfusive mice for 1 or 6 months blocks VaD-like neuropathological and functional outcomes. Single-cell transcriptomic profiling reveals that cerebral hypoperfusion induces numerous global changes in diverse brain cells including those of human AD brains. Remarkably, ~90% of these global changes are fully recovered with cis antibody, correlating with tau expression in cells. Thus, cis P-tau is a major common early driver of AD and VaD, and cis antibody has a potential role in the early detection, prevention and treatment of these devastating diseases.
Project description:We performed ribosome profiling in HEK293T cells with treatment with either C13-benzamidyl cycloheximide (hereafter "benzamide") or cycloheximide.
Project description:Mechanical unloading by ventricular assist devices (VAD) leads to significant gene-expression changes often summarized as reverse remodeling. However, little is known on individual transcriptome changes during VAD-support and its relationship to non-failing hearts (NF). In addition no data are available for the transcriptome regulation during non-pulsatile VAD-support. Therefore we analysed the gene-expression patterns of 30 paired samples from VAD-supported (including 8 non-pulsatile VADs) and 8 non-failing control hearts (NF) using the first total human genome-array available. Transmural myocardial samples were collected for RNA-isolation. RNA was isolated by commercial methods and processed according to chip-manufacturer recommendations. cRNA were hybridized on Affymetrix HG-U133 Plus 2.0 arrays, providing coverage of the whole human genome Array. Data was analyzed using Microarray Analysis Suite 5.0 (Affymetrix) and clustered by Expressionist software (Genedata). 352 transcripts were differentially regulated between samples from VAD-implantation and NF, whereas 510 were significantly regulated between VAD-transplantation and NF (paired t-test p<0.001, fold change >=1.6). Remarkably, only a minor fraction of 111 transcripts was regulated in heart failure (HF) and during VAD-support. Unsupervised hierarchical clustering of paired VAD- and NF-samples revealed separation of HF- and NF- samples, however individual differentiation of VAD-implantation and VAD-transplantation was not accomplished. Clustering of pulsatile and non-pulsatile VAD did not lead to robust separation of gene expression patterns. During VAD-support myocardial gene expression changes do not indicate reversal of the HF-phenotype, but reveal a distinct HF-related pattern. Transcriptome analysis of pulsatile and non-pulsatile VAD-supported hearts did not provide evidence for a pump-mode specific transcriptome pattern. Microarrays were used to elucidate the differences between non-failing control hearts and those, suffering from end-stage heart failure pre and post mechanical unloading.