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ERK1/2 signaling is necessary for definitive endoderm formation

ABSTRACT: Purpose: To compare gene expressions of undifferentiated cells and differentiated cells treated with basal media, basal media + FGF2, basal media + SU5402 and bsal media + ERKiII. Methods:For differentiation, hPSCs was set up 2 days before initiation of DE at Day 0 (D0). On D0, media was replaced with using RPMI/2%B27, Activin, CHIR99021, LY294002 +/- FGF2, SU5402 or TCS ERKi. The cells were harvested on D3. Cells were cultured in triplicates for each condition, total RNA extraction was performed and 1000 ng of purified RNA was sent for RNA-seq. Genes that are differentially expressed were analyzed by comparing RNA expression at different conditions. Results: Principal component analysis confirmed that our replicate samples clustered together, indicating good reproducibility. ACLY and ACLYF30 samples clustered close to each other but were non-overlapping, suggesting hish similarity in their cellular identities. Contrastingly, ACLYSU and ACLYERKi samples are found isolated from each other and also from undifferentiated hPSCs (UD), ACLY and ACLYF30 samples, indicating that the inhibition of FGFR or ERK1/2 had a huge impact on redirecting DE cell fate despite the presence of DE-inducing conditions. Conclusions: Blocking of FGFR with SU5402 and ERK2 signalling with ERKiII perturb cell cycle and cell fate specification even in the presence of definitive endoderm-inducing conditions.

ORGANISM(S): Homo sapiens

PROVIDER: GSE110806 | GEO | 2023/06/15

REPOSITORIES: GEO

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FGF2-regulated gene expression in cultured mouse spermatogonial stem cells

Project description:Transcriptional profiling of mouse spermatogonial stem cells (SSCs) comparing control untreated SSCs with SSCs with exogenous FGF2 withdrawn and FGFR inhibitor SU5402 supplemented (-F+S). Results provide insight into the mechanisms of FGF2-supported in vitro self-renewal of SSCs.

2010-08-01 | GSE21294 | GEO

FGF2-regulated gene expression in cultured mouse spermatogonial stem cells

2010-08-01 | E-GEOD-21294 | biostudies-arrayexpress

Expression data by ACLY knockdown

Project description:De novo lipogenesis is activated in most cancers. Several lipogenic enzymes are implicated in oncogenesis and represent potential cancer therapeutic targets. RNA interference-mediated depletion of ATP citrate lyase (ACLY), the enzyme that catalyzes the first step of de novo lipogenesis, leads to growth suppression in a subset of human cancer cells. Here we demonstrate the molecular basis and potential biomarkers for ACLY-targeting therapy. First, suppression of cancer cell growth by ACLY depletion involves down-regulation of fatty acid elongase ELOVL6 at the transcriptional level. Lipid profiling revealed that ACLY depletion alters fatty acid composition in triglyceride; increased palmitate and decreased longer fatty acids, in accordance with ELOVL6 down-regulation. Second, ACLY depletion increases reactive oxygen species (ROS), whereas addition of antioxidant reduces ROS and attenuates the growth suppression. Third, ACLY depletion or ROS stimulation induce phosphorylation of AMP-activated protein kinase (AMPK), a sensor of energy and lipid metabolism. Analysis of various cancer cell lines revealed that the levels of AMPK phosphorylation (p-AMPK) correlate with the basal ROS levels, and that cancer cells with low basal p-AMPK (i.e., low basal ROS) levels are highly susceptible to ACLY depletion-mediated growth suppression. Finally, in clinical colon cancer tissues, p-AMPK levels are significantly decreased in aggressive tumors and correlate with the levels of 8-hydroxydeoxyguanosine, a hallmark of ROS stimulation. Together, these data suggest that ACLY inhibition suppresses cancer growth via palmitate-mediated lipotoxicity, and p-AMPK could be a predictive biomarker for its therapeutic outcome. Two cell lines are treated with ACLY siRNA. The samples include controls of each cell line.

2014-04-13 | E-GEOD-37243 | biostudies-arrayexpress

Homo sapiens

Project description:Transcriptional profiling of primed hPSCs (KSR/FGF2 based media)

| PRJNA355894 | ENA

Expression data by ACLY knockdown

2014-04-13 | GSE37243 | GEO

Effect of FGF singaling inhibition on gene expression on limb bud mesenchyme explants.

Project description:To investigate the role FGF signaling in limb bud development we inhibited FGF signaling in limb bud explants by treatment with 5µM SU5402, an FGFR inhibitor, or DMSO as control. We then performed mRNA sequencing on RNA collected after 48h of treatment to analyse the effect on gene expression.

2023-08-24 | GSE223444 | GEO

Characterising the cellular activity of new Fibroblast Growth Factor Receptor tyrosine kinase inhibitors and identification of a gatekeeper mutation in FGFR3 as a mechanism of acquired resistance.

Project description:Fibroblast growth factor receptors (FGFRs) can act as driving oncoproteins in certain cancers due to mutation, over-expression or activating gene fusions and are therefore attractive drug targets. Here we have characterized tumour cell responses to three new inhibitors of FGFR1-3, AZ12576089, AZ12908010 and the clinical candidate AZD4547, making comparisons with the well-characterized FGFR inhibitor PD173074. Using a panel of 16 human tumour cell lines we show that the anti-proliferative activity of AZ12908010 and AZD4547 is strongly linked to the presence of de-regulated FGFR signalling. In contrast, AZ12576089 was also able to inhibit proliferation of cells lacking de-regulated FGFR, suggesting off-target effects. Acquired resistance to targeted tyrosine kinase inhibitors (TKIs) is a growing problem in the clinic. To assess how FGFR-dependent tumour cells may adapt to long-term exposure to FGFR inhibitors we generated a derivative of the KMS-11 myeloma cell line (FGFRY373C) with acquired resistance to AZ12908010 (KMS-11R cells). Basal P-FGFR3, P-FRS2 and P-ERK1/2 and D-type cyclins were all inhibited by AZ12908010 in parental KMS-11 cells whereas these markers were constitutively elevated and refractory to drug in KMS-11R cells. Sequencing of FGFR3 in KMS-11R cells revealed the presence of a heterozygous mutation at the gatekeeper residue, encoding FGFR3V555M. Consistent with this KMS-11R cells were cross-resistant to AZD4547 and PD173074 but remained fully sensitive to AZ12576089, confirming that the anti-proliferative effects of AZ12576089 are not related to FGFR inhibition. These results define the selectivity and efficacy of two new FGFR inhibitors and identify a secondary gatekeeper mutation as a mechanism of acquired resistance to FGFR inhibitors that should be anticipated as clinical evaluation proceeds.

2012-10-02 | E-TABM-1222 | biostudies-arrayexpress

Secreted footprint of pluripotency in Man-13 HeSCs

Project description:With a view to developing a much-needed non-invasive method for monitoring the healthy pluripotent state of human stem cells in culture, we undertook proteomic analysis of the waste medium from cultured induced (Rebl.PAT) human pluripotent stem cells (hPSCs). Cells were grown in E8 medium to maintain pluripotency, and then transferred to FGF2 and TGFβ deficient E6 media for 48 hours to replicate an early, undirected dissolution of pluripotency. We identified a distinct proteomic footprint associated with early loss of pluripotency in both hPSC lines, and a strong correlation with changes in the transcriptome. We demonstrate that multiplexing of four E8- against four E6- enriched secretome biomarkers provides a robust, diagnostic metric for pluripotent state. These biomarkers were further confirmed by Western blotting which demonstrated consistent correlation with the pluripotent state across cell lines, and in response to a recovery assay.

2024-03-26 | PXD045525 | Pride

Secreted footprint of pluripotency in REBL.PAT IPSCs

2024-03-26 | PXD045347 | Pride

Impact of ATP-citrate lyase catalytic activity and serine 455 phosphorylation on histone acetylation and inflammatory responses in human monocytic cells

Project description:ATP-citrate lyase (ACLY) is a key enzyme provoking metabolic and epigenetic gene regulation. Molecularly, these functions are exerted by the provision of acetyl-coenzyme A, which is then used as a substrate for de novo lipogenesis or as an acetyl-group donor in acetylation reactions. It has been demonstrated that ACLY activity can be positively regulated via phosphorylation at serine 455 by Akt and protein kinase A. Nonetheless, the impact of phosphorylation on ACLY function in human myeloid cells is poorly understood. In this study we reconstituted ACLY knockout human monocytic THP-1 cells with a wild type ACLY as well as catalytically inactive H760A, and phosphorylation-deficient S455A mutants. Using these cell lines, we determined the impact of ACLY activity and phosphorylation on histone acetylation and pro-inflammatory gene expression in response to lipopolysaccharide (LPS). Our results show that ACLY serine 455 phosphorylation does not influence the proper enzymatic function of ACLY, since both, wild type ACLY and phosphorylation-deficient mutant, exhibited increased cell growth and histone acetylation as compared to cells with a loss of ACLY activity. Transcriptome analysis revealed enhanced expression of pro-inflammatory and interferon response genes in ACLY knockout and H760A THP-1 cells under unstimulated or LPS-treated conditions. At the same time, S455A ACLY-expressing cells showed a phenotype very similar to wild type cells. Contrary to ACLY knockout, pharmacological inhibition of ACLY in THP-1 cells or in primary human macrophages does not enhance LPS-triggered pro-inflammatory gene expression. Our data thus suggest that ACLY retains functionality in the absence of Akt/PKA-mediated phosphorylation in human myeloid cells. Furthermore, loss of ACLY activity may elicit long-term adaptive mechanisms, increasing inflammatory responses.

2022-11-30 | GSE216003 | GEO

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