An autophagy CRISPR Screen in Jurkat cells
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE147488 | GEO | 2021/01/13
REPOSITORIES: GEO
Similar Datasets
Project description:An autophagy CRISPR Screen in Jurkat cells
| PRJNA614973 | ENA
Project description:Background: Abnormal autophagy and TGFβ-SMAD3/7 signaling pathway plays an important role in intrauterine adhesions (IUA); however, the exact underlying mechanisms remain unclear. In this study, we aimed to detect whether SMAD7 effected IUA via regulating autophagy and TGFβ-SMAD3 signaling pathway. Methods: The expression of p-SMAD3 and SMAD7 were detected by Immunohistochemistry. Endometrial fibrosis was detected by masson staining. The expression of protein related to autophagy and fibrosis was detected by western blot. The autophagic flux was monitored via Tandem mRFP-GFP-LC3 fluorescence system. Chip assay was used for SMAD3 binding site analysis. SMAD7 knockout mice were used to investigate the regulation of SMAD7 on intrauterine adhesions and autophagy. Results: we observed that patients with IUA exhibited a lower expression of SMAD7. In endometrial stromal cells, the silencing of SMAD7 inhibited autophagic flux, whereas overexpressed SMAD7 promoted autophagic flux. We also found that SMAD7-mediated autophagic flux regulated stromal-myofibroblast transition. These phenotypes are regulated by the transforming growth factor (TGF)β-SMAD3 signaling pathway. We found that SMAD3 directly binds to the 3ʹ-untranslated region of transcription factor EB (TFEB) and inhibits its transcription. SMAD7 promoted autophagic flux by inhibiting SMAD3, thereby promoting the expression of TFEB. The endometria of SMAD7 knockout mice showed a fibrotic phenotype. Simultaneously, autophagic flux was inhibited. On administering the autophagy activator rapamycin, endometrial fibrosis of SMAD7 gene conditional knockout mice was partially restored. Conclusions: The loss of SMAD7 promotes endometrial fibrosis by inhibited autophagic flux via the TGFβ-SMAD3 pathway. Therefore, this study reveals a potential therapeutic target for IUA.
2024-10-11 | GSE272824 | GEO
Project description:The aim of the project was to characterize changes in gene expression that are associated with induced autophagic flux. We engineered HeLa, HEK 293 and SH-SY5Y cell lines to express tandem-fluorecent LC3 (tf-LC3). The ratio of the red and green fluorophores indicated how much of the LC3 is in the acidic interior of lysosomes, which was a proxy measure for autophagic flux. RNA sequencing was performed for the cell lines at baseline and 1h, 15h and 30h after treatments. Additional untreated samples were also sequenced at some but not all time points. Autophagy was induced by amino acid starvation or mTOR inhibition (AZD8055).
2022-08-25 | E-MTAB-12020 | biostudies-arrayexpress
Project description:We isolated the human colon cancer cells expressing high leveles and low levels of autophagic activity by introdicing an autophagic flux indicator in organoids and performed RNA-seq analyses.
2023-04-23 | GSE193936 | GEO
Project description:Autophagy is an essential cellular process that is deeply integrated with innate immune signaling; however, studies that examine autophagy in the context of inflammatory conditions are lacking. Here, using mouse models with a constitutively active variant of the autophagy gene Beclin1, we show that increased autophagy dampens cytokine production in models of inflammation and adherent-invasive Escherichia coli (AIEC) infection. We further analyzed primary macrophages from these animals with a combination of transcriptomics and proteomics to identify novel mechanistic targets downstream of autophagy. Our study reveals glutamine metabolism and the RNF128/TBK1 axis as independent regulators of inflammation. Altogether, our work highlights increased autophagic flux as a potential approach to reduce inflammation and defines independent mechanistic cascades involved in this control.
2023-06-01 | GSE206124 | GEO
Project description:Autophagy is a catabolic pathway that maintains cellular homeostasis under various stress conditions, including nutrient-deprived conditions. To elevate autophagic flux to a sufficient level under stress conditions, transcriptional activation of autophagy genes occurs to replenish autophagy components. Here, using combination of RNA-seq, ATAC-seq and ChIP-seq, we demonstrated found that plant homeodomain finger protein 20 (Phf20PHF20), which is an epigenetic reader possessing methyl binding activity, plays a key role in controlling the expression of autophagy genes. PHF20 activates autophagy genes through enhancer activation via H3K36me2 binding activity as an epigenetic reader and that our findings emphasize the importance of nuclear regulation of autophagy.
2022-06-23 | GSE193392 | GEO
Project description:Autophagy is a catabolic pathway that maintains cellular homeostasis under various stress conditions, including nutrient-deprived conditions. To elevate autophagic flux to a sufficient level under stress conditions, transcriptional activation of autophagy genes occurs to replenish autophagy components. Here, using combination of RNA-seq, ATAC-seq and ChIP-seq, we demonstrated found that plant homeodomain finger protein 20 (Phf20PHF20), which is an epigenetic reader possessing methyl binding activity, plays a key role in controlling the expression of autophagy genes. PHF20 activates autophagy genes through enhancer activation via H3K36me2 binding activity as an epigenetic reader and that our findings emphasize the importance of nuclear regulation of autophagy.
2022-06-23 | GSE193217 | GEO
Project description:Autophagy is a catabolic pathway that maintains cellular homeostasis under various stress conditions, including nutrient-deprived conditions. To elevate autophagic flux to a sufficient level under stress conditions, transcriptional activation of autophagy genes occurs to replenish autophagy components. Here, using combination of RNA-seq, ATAC-seq and ChIP-seq, we demonstrated found that plant homeodomain finger protein 20 (Phf20PHF20), which is an epigenetic reader possessing methyl binding activity, plays a key role in controlling the expression of autophagy genes. PHF20 activates autophagy genes through enhancer activation via H3K36me2 binding activity as an epigenetic reader and that our findings emphasize the importance of nuclear regulation of autophagy.
2022-06-23 | GSE193215 | GEO
Project description:Macroautophagy (hereafter, autophagy) is a process that directs the degradation of cytoplasmic material in lysosomes. In addition to its homeostatic roles, autophagy undergoes dynamic positive and negative regulation in response to multiple forms of cellular stress, thus enabling the survival of cells. However, the precise mechanisms of autophagy regulation are not fully understood. To identify potential negative regulators of autophagy, we performed a genome-wide CRISPR screen using the quantitative autophagic flux reporter GFP-LC3-RFP. We identified phosphoribosylformylglycinamidine synthase (PFAS), a component of the de novo purine synthesis pathway, as one such negative regulator of autophagy. Autophagy was activated in cells lacking PFAS or phosphoribosyl pyrophosphate amidotransferase (PPAT), another de novo purine synthesis enzyme, or treated with methotrexate when exogenous levels of purines were insufficient. Purine starvation-induced autophagy activation was concomitant with mTORC1 suppression, and was profoundly suppressed in cells deficient for TSC2, which negatively regulates mTORC1 through inhibition of RHEB, suggesting that purines regulate autophagy through the TSC-RHEB-mTORC1 signaling axis. Moreover, depletion of the pyrimidine synthesis enzymes carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase (CAD) and dihydroorotate dehydrogenase (DHODH) activated autophagy as well, although mTORC1 activity was not altered by pyrimidine shortage. These results suggest a different mechanism of autophagy induction between purine and pyrimidine starvation. These findings provide novel insights into the regulation of autophagy by nucleotides and possibly the role of autophagy in nucleotide metabolism, leading to further developing anticancer strategies involving nucleotide synthesis and autophagy.
2021-05-11 | GSE164778 | GEO
Project description:Autophagy to apoptosis switching event is an unexplored area. We were interested to explore the gene expression profile at this juncture. Our Microarray data emphasized that among all eNOS and p62 genes were markedly induced. In fuctional level eNOS expression activates mTORC1 followed by inhibition of autophagy. This ultimately allowed accumulation of p62 . Intraccellular accumulation of p62 sequestered unfolded toxic protein aggregates in mitochondria and ER resulting in to impaired redox regulation. Intraccelular ROS generation further sensitized cells for apoptosis and tilted autophagic response to apoptosis. Cells were treated with Tunicamycin as an inducer of both autophagy and apoptosis. At early stage of Tunicamycin treatment 24h , prostate cancer cell PC3 showed activation of autophagic process where apoptosis was not evident. Sustained ER stress with Tunicamycin induced late apoptoisis at 72h of treatment. Hence we isolated total RNA from Untreated cells, cells with Tunicamycin treatment after 24h and 72h. Further the RNA were used to perform whole genome microarray to analyze the gene expression profile at autophagy and apoptosis juncture. The selected genes were also validated by qPCR and western bot. Morover RNAi study were implemented to evaluate the signaling crosstalk at the juncture of autophagy and apoptosis.
2014-05-22 | E-GEOD-38643 | biostudies-arrayexpress