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Next Generation Sequencing Facilitates Quantitative Analysis of Transcriptomes at LncRNA and mRNA and miRNA levels between Hep2 cells with stable transfection of MYCT1 and non-transfection of MYCT1

ABSTRACT: Next Generation Sequencing Facilitates Quantitative Analysis of Transcriptomes at LncRNA and mRNA and miRNA levels between Hep2 cells with stable transfection of MYCT1 and non-transfection of MYCT1

PROVIDER: PRJNA507997 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR8272056_1.fastq.gz	Fastqsanger.gz
	SRR8272056_2.fastq.gz	Fastqsanger.gz
	SRR8272057_1.fastq.gz	Fastqsanger.gz
	SRR8272057_2.fastq.gz	Fastqsanger.gz
	SRR8272058.fastq.gz	Fastqsanger.gz

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Next Generation Sequencing Facilitates Quantitative Analysis of Transcriptomes at LncRNA and mRNA and miRNA levels between Hep2 cells with stable transfection of MYCT1 and non-transfection of MYCT1

Project description:Purpose: To obtain the differentially expressed LncRNA, mRNAs and miRNAs between the two groups of cells. Results: We identified 174 LncRNAs, 327 mRNAs and 235 miRNAs differentially expressed in MYCT1-overexpressed Hep2 cells compared to the controls.

2018-12-04 | GSE123275 | GEO

MYCT1 CONTROLS ENVIRONMENTAL SENSING IN HUMAN HEMATOPOIETIC STEM CELLS

Project description:Hematopoietic stem cells (HSC) rely on a unique regulatory machinery that facilitates life-long blood production and enables reconstitution of the entire hematopoietic system upon transplantation. However, the biological processes governing human HSC self-renewal and engraftment ability are poorly understood and challenging to recapitulate ex vivo to facilitate robust human HSC expansion. We discovered a novel HSC regulatory protein, MYCT1 (MYCT target 1), that is selectively expressed in endothelial cells (EC) and undifferentiated human HSPCs but becomes drastically downregulated during HSC culture. Lentiviral knockdown of MYCT1 in human foetal liver and cord blood HSPCs revealed a critical role for MYCT1 in governing human HSPC expansion and engraftment ability. Single cell RNAseq of human CB HSPCs after MYCT1 knockdown and overexpression revealed that MYCT1 governs HSC functional competence and modulates cellular properties essential for HSC stemness, such as low mitochondrial metabolic activity. Indeed, restoring the compromised MYCT1 expression in cultured human CB HSPCs improved ex vivo expansion of the most undifferentiated human HSPCs and enhanced their engraftment ability. We found that MYCT1 is localized in the endosomal membrane and interacts with vesicle trafficking regulators and signalling machinery essential for HSC and EC function. Loss of MYCT1 led to excessive endocytosis and hyperactive signalling responses to cytokines, whereas restoring MYCT1 expression in cultured CB HSPCs balanced the abnormal endocytosis associated with prolonged culture and fine-tuned signalling responses. Our work identifies MYCT1-moderated endocytosis and environmental sensing as an essential regulatory mechanism required to preserve human HSC stemness, and pinpoints silencing of MYCT1 as a critical contributor to the dysfunction of cultured human HSCs that needs to be addressed to improve human HSC culture strategies.

2024-04-15 | PXD042257 | Pride

MYCT1 Controls Environmental Sensing in human Hematopoietic Stem Cells [scRNA-seq]

Project description:We identified a novel HSC regulatory gene, MYCT1 (MYC target 1), that is selectively expressed in undifferentiated human HSPC and is critical for human HSC expansion and engraftment. MYCT1 knockdown (KD) in human fetal liver and cord blood (CB) HSPCs impaired expansion ex vivo and engraftment after transplantation, whereas restoring the compromised MYCT1 expression via lentiviral overexpression (OE) in cultured human CB HSPCs improved ex vivo expansion of the most undifferentiated human HSPCs (CD34+CD38-CD90+CD45RA-EPCR+ITGA3+) and enhanced their engraftment ability. We performed single cell RNAseq of uncultured human CB HSPCs, as well as 72 hours after MYCT1 KD and OE to identify MYCT1-regulated programs in HSC and understand the role of MYCT1 in governing human HSPC expansion and engraftment ability. Functional enrichment analysis linked the loss of MYCT1 in HLF+ HSCs to a profound dysregulation of multiple cellular programs essential for HSC stemness, such as oxidative phosphorylation or proteostasis, while MYCT1 OE had the opposite effect and showed closer similarity to uncultured HLF+ HSCs. These data indicate that the loss or gain of MYCT1 expression elicits major effects on programs regulating human HSC functional competence.

2024-04-08 | GSE232361 | GEO

MYCT1 Controls Environmental Sensing in human Hematopoietic Stem Cells [scRNA-seq Dextran]

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MYCT1 Controls Environmental Sensing in human Hematopoietic Stem Cells [RNA-seq]

Project description:We identified a novel HSC (hematopoietic stem cell) regulatory gene, MYCT1 (MYC target 1), that is selectively expressed in undifferentiated human HSPC and is critical for human HSC expansion and engraftment. To define the molecular function of MYCT1 we performed cell fractionation, immunofluorescence, and immunoprecipitation coupled with high sensitivity mass spectrometry of V5-tagged MYCT1 in KG1 hematopoietic cells and human endothelial cells (E4-immortalized HUVEC, E4EC (Butler et al. 2012)). We found that MYCT1 localizes in the endosomal membrane and interacts with vesicle trafficking regulators and signalling machinery essential for HSC and endothelial cell function. We performed RNAseq to asses the expression of the identified MYCT1 interactors in KG1 and E4EC cell lines.

2024-04-08 | GSE232360 | GEO

Single Cell RNA Sequencing of WT and Cdh5-cre Myct1 fl/fl Endothelium

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Transfection with Glucuronidase A affects general proteome pattern in Giardia lamblia trophozoites

Project description:Giardia lamblia is an important causative agent of persistent diarrhea in humans, domestic animals, and cattle. Basic research is usually performed with the strain WBC6 and includes genetic manipulations such as transfections. Here, we investigate how transfection with a plasmid causing stable expression of a foreign gene affects the whole proteome pattern. Using shotgun mass spectrometry, we compare the proteomes of untransfected trophozoites to trophozoites transfected with Escherichia coli glucuronidase A (GusA). Besides GusA, which is detected in the transfected trophozoites only, the proteomes of untransfected and transfected trophozoites differ by 132 differentially expressed proteins. In particular, transfection induces antigenic variation. Since transfection causing stable expression affects the proteome pattern, transfection experiments should take into account this effect. Due to a unique peptide panel, GusA is an example for a suitable internal standard for experiments involving transfected cells.

2021-01-07 | PXD022565 | Pride

EWS-FLI1-transfection of HEK293

Project description:Response of HEK293-cells after transfection with EWS-FLI1. HEK293 cells were transfected with the expression vector pIRES2-EGFP containing type I EWS-FLI1 or empty control vector. For transient transfection standard DEAE dextran method was used and RNA was isolated 48h post transfection. For stable transfection cells were transfected using FuGENE 6 (Roche, Mannheim, Germany) and cells were selected with 400 ug/mL G418. DNA-microarray analysis was performed using Affymetrix HG-U133A microarrays.(see Staege et al. Cancer Res. 2004) Keywords = HEK293 Keywords = EWS-FLI1 Keywords = Ewing family tumors Keywords: other

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Proteomic analysis of HeLa cells after stable transfection with the Chlamydia trachomatis CT143 gene

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