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  • Activation of JAK-STAT3 signaling by leukemia inhibitory factor (LIF) is required for maintaining self-renewal of mouse embryonic stem cells (mESCs). STAT3 perform cell type-specific roles in different cell type, here we revisit the role of STAT3 using mouse female germ stem cell (mFGSCs). We applied CRISPR/Cas9 system to generate Stat3 knockout FGSCs and then observed cell growth inhibition and cell cycle arrest in KO cell line. By combining genome wide ChIP-Seq and RNA-Seq, we identified 5990 STAT3 binding sites and discovered serval genes specific regulated by STAT3 that were involved in stem cell proliferation and female gonad development in FGSCs. In general, we identify key roles of STAT3 for sustains self-renewal and proliferation for FGSCs in this study.
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  • Activation of JAK-STAT3 signaling by leukemia inhibitory factor (LIF) is required for maintaining self-renewal of mouse embryonic stem cells (mESCs). STAT3 perform cell type-specific roles in different cell type, here we revisit the role of STAT3 using mouse female germ stem cell (mFGSCs). We applied CRISPR/Cas9 system to generate Stat3 knockout FGSCs and then observed cell growth inhibition and cell cycle arrest in KO cell line. By combining genome wide ChIP-Seq and RNA-Seq, we identified 5990 STAT3 binding sites and discovered serval genes specific regulated by STAT3 that were involved in stem cell proliferation and female gonad development in FGSCs. In general, we identify key roles of STAT3 for sustains self-renewal and proliferation for FGSCs in this study.
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  • Immune checkpoint blockade has revolutionized cancer therapy. In particular, inhibition of programmed cell death protein 1 (PD-1) is effective for the treatment of metastatic melanoma and other cancers. Despite a dramatic increase in progression-free survival, a large proportion of patients do not show durable response. Therefore, predictive biomarkers of clinical response are urgently needed. Here, we employed high-dimensional single cell mass cytometry and a bioinformatics pipeline for the in-depth characterization of the immune cell subsets in the peripheral blood of metastatic melanoma patients before and after anti-PD-1 immunotherapy. During therapy, we observed a clear treatment response to immunotherapy in the T cell compartment. However, prior to commending therapy a strong predictor of progression free and overall survival in response to anti-PD-1 immunotherapy was the frequency of CD14+CD16-HLA-DRhi monocytes. We could confirm this by conventional flow cytometry in an independent validation cohort and propose this as a novel predictive biomarker for therapy decisions in the clinic. In order to determine whether there are cell intrinsic changes in the monocyte signature, we performed RNA sequencing on sorted CD14+CD16-HLA-DRhi cells from HD, NR and R at baseline. Representative samples (n=4, each) of responders/non responders/ and healthy donors were selected from archival samples stored in the dermatology biobank according to the same clinical criteria used in the discovery and validation cohorts for CyTOF and FACS analysis. CD14+CD16-HLA-DRhiLin- (CD3, CD4, CD19, CD45RO) monocytes were sorted from frozen PBMC form blood samples from HD, R and NR at baseline.
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  • To understand the regulation of cytotoxicity by decidual CD8+ T cells (CD8+ dT) at the maternal-fetal interface, gene expression analysis of CCR7-CD45RA- effector-memory CD8+ T cells isolated from peripheral blood of unrelated healthy controls and decidual tissue from first trimester (6-12 weeks) and term (>37 weeks) pregnancy was performed. Furthermore, first trimester decidual effector-memory CD8+ T cells were stimulated with anti-CD3/CD28 in the presence of IL-2 for 12 hours or 72 hours. RNA was isolated and gene expression profiles were generated by employing Affymetrix HG_U133_Plus 2 arrays on the Affymetrix Geneatlas system.
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  • Four purified RNA extracts (2 from cells grown in glucose supplemented medium and another 2 from cells grown in PE supplemented medium) were subjected to sequencing. Following cDNA library preparations, the final sequencing libraries were quantified using the KAPA kit (KAPA Biosystem, USA) on a Stratagene Mx-3005P qPCR system (Agilent Technologies, USA) and the respective library sizes were confirmed using Agilent Bioanalyzer High Sensitivity DNA Chip (Agilent Technologies, USA). The resulting libraries were subjected to cluster generation and sequenced using an Illumina flow cell, 202 cycles (101 bp paired-end reads) on the Illumina HiSeq 2000 system (Illumina, USA).
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  • Cardiomyocytes derived from human pluripotent stem cells were exposed to the cardiotoxic drug Doxorubicin in order to assess the utility of this cell system as a model for drug-induced cardiotoxicity. Cells are exposed to different concentrations of doxorubicin for up to 48 hours followed by a 12 days recovery period.
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  • Cutaneous squamous cell carcinoma (cSCC) is the second most common skin cancer type and arises from keratinocytes. Most cSCC progress from a UV-induced precancerous lesion termed actinic keratosis (AK). Despite various efforts to characterize these lesions molecularly, the etiology of AK and its progression to cSCC remain only partially understood. Here we have used Infinium MethylationEPIC BeadChips to interrogate the DNA methylation status of about 850.000 CpGs in epidermal preparations from healthy skin, AK and cSCC. Importantly, we found that the premalignant AK samples displayed classical features of cancer methylomes and were highly similar to cSCC methylomes. Further analysis identified typical features of stem cell methylomes, such as a reduced DNA methylation age, non-CpG methylation and stem cell-related keratin and enhancer methylation patterns. Interestingly, this signature was detected only in one half of the AK and cSCC samples, while the other half showed methylation patterns that were more closely related to the control epidermis. These findings suggest the existence of two distinct subclasses of AK and cSCC that originate from distinct keratinocyte differentiation stages.
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  • The mature leaves (full-expanded leaves) and developing leaves (30%length of full-expanded) of 8 species of the genus Flaveria (F.pringlei, F.robusta, F.floridana, F.ramosissima, F.brownii, F.palmeri, F.bidentis and F.trinervia) Transcriptome analysis. Plants were grown in the growth chamber (12hr light - 12hr dark, 24℃, 200-300umol m-2 s-1). Total RNA was isolated from the youngest full expanded leaves or developing leaves (30%length of full-expanded) of each species.
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  • To monitor the global changes in gene expression of Staphylococcus aureus USA300 TCH1516 under hypochlorite stress by RNA-seq, cultivation was performed in Luria Bertani (LB) medium in triplicate at 37C until cells have reached an optical density at 540nm of 2.0. Cells were harvested by centrifugation, washed with Belitsky minimal medium (BMM) and adapted to BMM for one hour before exposure to 150 M NaOCl stress. S. aureus cells of 3 replicate experiments were harvested before and 30 min after exposure to 150 M NaOCl and disrupted in 3 mM EDTA/ 200 mM NaCl lysis buffer with a Precellys24 Ribolyzer. RNA isolation was performed using the phenol-chloroform-isoamylalcohol approach. After precipitation with 3 M sodium acetate and isopropanol, the total RNA was washed with cold ethanol and the pellet was solved in sterile water. Initially RNA quality was checked by Trinean Xpose (Gentbrugge,Belgium) and Agilent RNA Nano 6000 kit on Agilent 2100 Bioanalyzer (Agilent Technologies, Bblingen, Germany). Samples contaminated with DNA were treated with DNase (Qiagen), cleaned as described above and rechecked by Xpose and Agilent Bioanalyzer. Finally RNA was free of DNA with an RNA Integrity Number (RIN) > 9 and rRNA Ratio [23s / 16s] > 1.5. Ribo-Zero rRNA Removal Kit (Bacteria) from Illumina (San Diego, CA, USA) was used to remove the ribosomal RNA molecules from the isolated total RNA. Removal of rRNA was checked by Agilent RNA Pico 6000 kit on Agilent 2100 Bioanalyzer (Agilent Technologies, Bblingen, Germany). RNA was free of detectable rRNA. TruSeq Stranded mRNA Library Prep Kit from Illumina (San Diego, CA, USA) was used to prepare cDNA libraries. The resulting cDNAs were sequenced paired end on an Illumina HiSeq 1500 and MiSeq system (San Diego, CA, USA) using 50 and 75 bp read length, respectively.
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  • A549 cells were treated for 8 or 20 h using 20 ng/mL brefeldin A (BFA), 5 µM golgicide A (GCA), 10 µM monensin (MON) or the appropriate vehicle control (BFA/EtOH, MON/EtOH, GCA/DMSO).
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