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ABSTRACT:
PROVIDER: PRJNA701836 | ENA |
REPOSITORIES: ENA
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Project description:We examined the effect of nicotinamide on cell survival and differentiation in human pluripotent stem cells. Nicotinamide inhibited the phosphorylation of myosin light chain, suppressed actomyosin contraction, and led to improved cell survival after individualization. Then we analyzed the global gene expression profile after 24 hours of nicotinamide and ROCK inhibitor treatment, and found that the gene expression profile of human embryonic stem cell (hESC) treated with nicotinamide was much different from that of ROCK inhibitor treatment. Further analysis demonstrates that nicotinamide is an inhibitor of multiple kinases, including ROCK and CK1. We demonstrated that nicotinamide affected human embryonic stem cell (hESC) pluripotency and differentiation as a selective kinase inhibitor.
2018-10-29 | GSE121230 | GEO
Project description:Background: Naïve human embryonic stem cells (hESCs) have been isolated that more closely resemble the pre-implantation epiblast compared to conventional “primed” hESCs, but the signaling principles underlying these discrete stem cell states remain incompletely understood. Methods:Here we performed high-throughput screening using a library of >3,000 well-annotated compounds to identify essential signaling requirements for naïve human pluripotency. Results:We report that MEK1/2 inhibitors can be replaced during maintenance of naïve human pluripotency by inhibitors targeting either upstream (FGFR, RAF1) or downstream (ERK1/2) kinases. Naïve hESCs maintained under these alternative conditions display elevated levels of ERK phosphorylation but retain genome-wide DNA hypomethylation and a transcriptional identity of the pre-implantation epiblast. In contrast, dual inhibition of MEK and ERK promotes efficient primed-to-naïve resetting in combination with PKC, ROCK, and TNKS inhibitors and Activin A. Conclusions: This work demonstrates that induction and maintenance of naïve human pluripotency are governed by distinct signaling requirements.
2021-06-15 | GSE153212 | GEO
Project description:Background: Naïve human embryonic stem cells (hESCs) have been isolated that more closely resemble the pre-implantation epiblast compared to conventional “primed” hESCs, but the signaling principles underlying these discrete stem cell states remain incompletely understood. Methods:Here we performed high-throughput screening using a library of >3,000 well-annotated compounds to identify essential signaling requirements for naïve human pluripotency. Results:We report that MEK1/2 inhibitors can be replaced during maintenance of naïve human pluripotency by inhibitors targeting either upstream (FGFR, RAF1) or downstream (ERK1/2) kinases. Naïve hESCs maintained under these alternative conditions display elevated levels of ERK phosphorylation but retain genome-wide DNA hypomethylation and a transcriptional identity of the pre-implantation epiblast. In contrast, dual inhibition of MEK and ERK promotes efficient primed-to-naïve resetting in combination with PKC, ROCK, and TNKS inhibitors and Activin A. Conclusions: This work demonstrates that induction and maintenance of naïve human pluripotency are governed by distinct signaling requirements.
2021-06-15 | GSE153213 | GEO
Project description:The Casein Kinase 1 (CK1) family of Ser/Thr protein kinases are implicated in the regulation of many cellular processes, including cell cycle, circadian rhythm, Wnt and Sonic Hedgehog signalling. Regarded as constitutively active kinases, their regulation in cells is critically important but poorly understood. We report here that members of the FAM83 family of uncharacterized proteins are central regulators of CK1 isoforms in cells. The eight members of the FAM83 family (A-H) interact and co-localize with different CK1 alpha, alpha-like, delta and epsilon isoforms. We demonstrate that the interaction with CK1 isoforms is mediated through a number of residues within a conserved domain of unknown function, termed DUF1669, which characterises the FAM83 family. CK1-binding deficient mutants of FAM83 proteins interfere with the subcellular localization of CK1 isoforms as well as FAM83 proteins themselves and their cellular functions. We propose that DUF1669 be renamed the Polypeptide Anchor of CK1 (PACK1) domain.
2018-05-24 | PXD009335 | Pride
Project description:Although substantial progress has been made in the treatment of B-cell acute lymphoblastic leukemia (B-ALL), the prognosis of patients with refractory or relapsed B-ALL remains dismal. Novel therapeutic strategies are needed to improve the outcome of these patients. KPT-9274 is a novel dual inhibitor of p21-activated kinase 4 (PAK4) and nicotinamide phosphoribosyltransferase (NAMPT). PAK4 is a serine/threonine kinases and it regulates a variety of protein kinases involved in cell survival, motility and proliferation. NAMPT is a rate-limiting enzyme in the salvage biosynthesis pathway of nicotinamide adenine dinucleotide (NAD), which plays a vital role in energy metabolism. Here, we show that KPT-9274 strongly inhibits B-ALL cell growth regardless of cytogenetic abnormalities. We also demonstrate the potent in vivo efficacy and tolerability of KPT-9274 in orthotopic xenograft murine models using patient-derived BALL cells. Although KPT-9274 affected both PAK4 signaling pathways and NAD+ dependent pathways, B-ALL cell growth inhibition mediated by KPT-9274 was largely abolished by nicotinic acid supplementation, indicating that the inhibitory effect of KPT- 9274 on B-ALL cell growth was mainly exerted by NAD+ depletion through blockade of NAMPT enzyme activity. Moreover, we found that B-ALL cells were especially vulnerable to NAD+ depletion, and the susceptibility to treatment with KPT-9274 was related to the reduced NAD+ reserve in B-ALL cells. NAD+ depletion may be a promising alternative approach to treating patients with B-ALL.
2017-05-01 | GSE86871 | GEO
Project description:To understand how mechanical cues regulate cell metabolism, we compared early changes in gene expression in Ras-transformed MCF10AtK1 mammary epithelial cells grown in high cytoskeletal tension conditions (plastics) vs. cells grown in low cytoskeletal tension conditions (inhibition of the non-muscle myosin II regulatory kinases ROCK and MLCK with standard small-molecule inhibitors Y27632 and ML7, respectively). Keywords: Expression profiling by array
2019-02-05 | GSE107275 | GEO
Project description:In this study, we examine the transcriptome of bovine TSCs under self-renewal that is sustained by inhibiting RhoA-Rock pathway. Using systems biology of spontaneous differentiation, we identify that TGFB1 induces differentiation of bovine TSCs. We analyse the transcriptome of TSCs under TGFB1 induced differentiation. We also analyze pathways of differentiation that are dependent and independent of RhoA-Rock signaling through analysis of the transcriptome of TSCs exposed to both TGFB1 and ROCK inhibition.
2022-03-11 | GSE181252 | GEO
Project description:Human embryonic stem cell derived retinal pigmented epithelial cells (hESC-RPE) are in clinical trials for the treatment of macular diseases. Currently, these cells take over three months to derive and subsequent months to mature and characterize. After only five to six passages the cells begin to undergo an epithelial-to-mesenchymal transition and are unsuitable for cellular therapies. We describe a novel passaging protocol and show that inhibition of Rho-associated, coiled coil containing protein kinases (ROCK1 and ROCK2) using Y-27632, allows extended passage of hESC-RPE in serum-free culture with maintenance of the RPE phenotype. After 30 population doublings, hESC-RPE at passage 13 maintain normal karyotype, display typical, polarized epithelial morphology, and continue to express RPE-specific genes. Passage 13 hESC-RPE show protein localization patterns similar to passage 2 cells, and display similar levels of growth factor secretion and phagocytosis of photoreceptor outer segments. Microarray analysis from day 2 cells shows several key pathways are altered by ROCK inhibition, including stimulation of the cell cycle and suppression of TGFM-NM-2 and Wnt signaling. These findings describe a means to greatly increase the yield of functional hESC-RPE for use in research and clinical trials. Two-condition experiment, Control vs Y-27632 treated hESC-RPE passage 5. Biological replicates: 4 control, 4 Y-27632. The experiments were technically carried out as dual channel (eg, Cy3 and Cy5-labeled samples hybridized to the same array) but processed as though they are single channel (Cy3 and Cy5 signals are calculated; Cy3/Cy5 ratios are not calculated). Therefore, there are 4 raw data files for total 8 samples.
2014-06-30 | E-GEOD-56618 | biostudies-arrayexpress
Project description:CK1 enzymes are conserved, acidophilic serine/threonine kinases with a variety of critical cellular functions; their misregulation contributes to cancer, neurodegenerative diseases, and sleep phase disorders. Here, we describe a new mechanism of CK1 regulation conserved from yeast to human – autophosphorylation of a threonine in the mobile L-EF loop proximal to the active site – that inhibits kinase activity. Consequently, yeast and human CK1 enzymes with phosphoablating mutations at this site are hyperactive in vitro. We used quantitative phosphoproteomics to show that disruption of this regulatory mechanism rewires CK1 signaling in Schizosaccharomyces pombe. In accord, we found that a known CK1 pathway, a mitotic checkpoint, is downregulated in these mutants, while new pathways that confer heat shock resistance and suppress meiotic transcripts are upregulated. Molecular dynamics simulations demonstrated that phosphorylation on the L-EF loop alters the conformation of the substrate docking site, and we propose that this affects which CK1 substrates can be phosphorylated. Due to the functional importance of the L-EF loop, which is unique to the CK1 family of kinases, this mechanism is likely to regulate the majority of CK1 enzymes in vivo.
2023-07-20 | PXD026766 | Pride
Project description:The goal of this experiment is to identify genes differentially expressed in MEF cell lines with a targeted MDMX mutation that prevents binding and inhibition of CK1-alpha kinase.
2020-04-07 | GSE148138 | GEO