Project description:Mitochondria play a key role in metabolic transitions involved in the reprogramming of somatic cells into induced pluripotent stem cells (iPSCs), but the underlying molecular mechanisms remain largely unexplored. To obtain new insight into the mechanisms of cellular reprogramming, we studied the role of FAH domain-containing protein 1 (FAHD1) in the reprogramming of murine embryonic fibroblasts (MEFs) into iPSCs and their subsequent differentiation into neuronal cells. MEFs from wild type (WT) and Fahd1-knock-out (KO) mice were reprogrammed into iPSCs and characterized for alterations in metabolic parameters and the expression of marker genes indicating mitochondrial biogenesis. Fahd1-KO MEFs showed a higher reprogramming efficiency accompanied by a significant increase in glycolytic activity as compared to WT. We also observed a strong increase of mitochondrial DNA copy number and expression of biogenesis marker genes in Fahd1-KO iPSCs relative to WT. Neuronal differentiation of iPSCs was accompanied by increased expression of mitochondrial biogenesis genes in both WT and Fahd1-KO neurons with higher expression in Fahd1-KO neurons. Together these observations establish a role of FAHD1 as a potential negative regulator of reprogramming and add additional insight into mechanisms by which FAHD1 modulates mitochondrial functions.
Project description:Ferroptosis is a type of programmed cell death distinct from apoptosis and necroptosis that plays an essential role in pathophysiological conditions such as neurodegenerative diseases and tumorigenesis. Massive lipid oxidation in an iron-dependent manner is a hallmark of ferroptosis.This modality of cell death is also characterized by perturbation of several metabolic pathways, predominantly fatty acid metabolism, thiol metabolism, iron homeostasis and the mevalonate pathway. We aimed to acquire data from different timepoints of ferroptotic death in order to get information about the primary and delayed phases of the ferroptotic response. For this purpose, we used model Pfa1 cells, which are 4-OH-TAM-inducible Gpx4-/- mouse immortalized fibroblasts [1]. GPX4 is one of the main intracellular ferroptosis regulators and inhibiting it is a classic approach to induce ferroptosis. Measuring protein fold changes at different ferroptotic stages and in nontreated Pfa1 cells could give useful information on the activation of genes involved in ferroptosis and non-genomic protein regulation during ferroptotic progression. Bottom-up proteomic data were acquired from samples obtained 24 and 48 hours after genetic induction of ferroptosis. Chromato-mass spectra were registered in DDA mode and are suitable for further label-free quantification. These data might be a valuable proteome basis for further investigation of ferroptosis and complement other available omics.
Project description:In the present study, we report proteome data of mouse embryonic fibroblasts (MEF cells) and immortalized mouse embryonic fibroblasts which are 4-OH-TAM-inducible Gpx4−/− (Pfa1 cells). Ferroptosis in Pfa1 cells was induced with Tamoxifen (Tam) addition and three timepoints were collected: 0h, 24h and 48h. Wild-type MEF cells were also measured as an alternative control without Tam addition. Raw data for HEK293 cells are provided as an independent quality control. MEF and Pfa1 cells (both kind gift from Marcus Conrad, Munich) were cultured in RPMI-1640 media (2 g/L glucose, 10% FBS, 2 mM Gibco GlutaMAX Supplement, 1% pen/strep) at 37C with 5% CO2 in a humidified incubator. Cells (300k) were seeded on Corning 100 mm tissue-culture treated culture dishes and incubated overnight. On the next day Pfa1 cells were treated with or without 1 µM Tam.
Project description:BackgroundMorphometric quantification of subtle craniofacial variation in studies of experimentally modified embryonic mice has proved valuable in determining the effects of developmental perturbations on craniofacial morphogenesis. The direct comparison of landmark coordinate data from embryos of many different mouse strains and mouse models can advance our understanding of the bases for craniofacial variation. We propose a standard set of craniofacial surface landmarks, for use with embryonic day (E) 10.5-12.5 mice, to serve as the foundation for this type of data compilation and analysis. We quantify the intra- and inter-observer landmark placement variation associated with each landmark and determine how the results of a simple ontogenetic analysis might be influenced by selection of landmark set.ResultsIntraobserver landmark placement error for experienced landmarkers generally remains below 0.1 mm, with some landmarks exhibiting higher values at E11.5 and E12.5. Interobserver error tends to increase with embryonic age and those landmarks defined on wide inflections of curves or facial processes exhibit the highest error. Landmarks with highest intra- or inter-observer are identified and we determine that their removal from the dataset does not significantly change the vectors of craniofacial shape change associated with an ontogenetic regression.ConclusionsOur quantification of landmark placement error demonstrates that it is preferable for a single observer to identify all landmark coordinates within a single study and that significant training and experience are necessary before a landmarker can produce data for use in larger meta-analyses. However, we are confident that this standard landmark set, once landmarks with higher error are removed, can serve as a foundation for a comparative dataset of facial morphogenesis across various mouse populations to help identify the developmental bases for phenotypic variation in the craniofacial complex.
Project description:We previously reported that partial disruption of the Ankrd26 gene in mice leads to hyperphagia and leptin-resistant obesity. To determine whether the Ankrd26 mutation can affect the development of adipocytes, we studied mouse embryo fibroblasts (MEFs) from the mutant mice. We found that Ankrd26(-/-) MEFs have a higher rate of spontaneous adipogenesis than normal MEFs and that adipocyte formation is greatly increased when the cells are induced with troglitazone alone or with a mixture of troglitazone, insulin, dexamethasone, and methylisobutylxanthine. Increased adipogenesis was detected as an increase in lipid droplet formation and in the expression of several markers of adipogenesis. There was an increase in expression of early stage adipogenesis genes such as Krox20, KLF5, C/EBPβ, C/EBPδ, and late stage adipogenesis regulators KLF15, C/EBPα, PPARγ, and aP2. There was also an increase in adipocyte stem cell markers CD34 and Sca-1 and preadipocyte markers Gata2 and Pref-1, indicating an increase in both stem cells and progenitor cells in the mutant MEFs. Furthermore, ERK was found constitutively activated in Anrd26(-/-) MEFs, and the addition of MEK inhibitors to mutant cells blocked ERK activation, decreased adipogenesis induction, and significantly reduced expression of C/EBPδ, KLF15, PPARγ2, CD34, and Pref-1 genes. We conclude that Ankrd26 gene disruption promotes adipocyte differentiation at both the progenitor commitment and differentiation steps and that ERK activation plays a role in this process.
Project description:Analysis of embryonic fibroblasts from GFP reporter mice indicates that the fibroblast cell type harbors a large collection of developmentally and phenotypically heterogeneous subtypes. Some of these cells exhibit multipotency, whereas others do not. Multiparameter flow cytometry analysis shows that a large number of distinct populations of fibroblast-like cells can be found in cultures initiated from different embryonic organs, and cells sorted according to their surface phenotype typically retain their characteristics on continued propagation in culture. Similarly, surface phenotypes of individual cloned fibroblast-like cells exhibit significant variation. The fibroblast cell class appears to contain a very large number of denumerable subtypes.