ABSTRACT: Mass spectrometry DIA analysis of MG132 treated Xenopus laevis egg extracts and compared with control (untreated) extracts to identify changes in protein levels upon MG132 treatment.
Project description:Yap (Yes-associated protein 1) is a downstream effector of the Hippo signaling pathway and known as a transcriptional co-activator. We previously identified Yap1 as a novel factor implicated in the control of the temporal DNA replication program in Xenopus retinal stem cells. However, whether Yap is directly involved in DNA replication dynamics and whether it could regulate DNA replication during early embryonic divisions in the absence of transcription remained to be investigated. We now found that in Yap depleted Xenopus laevis egg extracts the activation of replication origins was increased. In order to understand by which mechanism Yap could regulate the initiation of DNA replication in the absence of transcription we searched for new interacting factors. We immunoprecipitated Yap from Xenopus laevis egg extracts using anti-Yap, compared to control antibodies, and analyzed co-immunoprecipitating proteins by nanoLC/MS/MS.
Project description:Bone marrow stromal cells (BMSCs) are multipotent stem cells that preferentially differentiate into mesenchymal cells. If they can be dedifferentiated into embryonic stem cell-like cells, they will be a highly attractive source for cell therapy. Cell and egg extracts have been used in a few studies to evaluate nuclear reprogramming, but these have not examined cell pluripotency in any detail. In this study, we used a cell reversible permeabilization method to treat BMSC with Xenopus laevis mitotic egg extract. We observed an upregulation of the pluripotent protein Oct3/4 in BMSCs treated by this extract. We also further evaluated transcriptional changes with a focused stem cell oligonucleotide array. A number of genes involved in the Notch or Wnt signaling pathways were upregulated in BMSC exposed to Xenopus egg extract. In conclusion, our microarray data from BMSCs exposure to egg extracts may provide interesting clues regarding factors involved in nuclear reprogramming. Our approach is an alternative method towards dedifferentiation of cells without genetic modification, which is preferable in the clinical situation. Keywords: Cell type comparison
Project description:The aim of the project was to analyse the role of DONSON during DNA replication reaction in Xenopus laevis egg extract. To test what is the impact of DONSON on replication we decided to remove DONSON from egg extract by immunodepletion and analyse its impact on DNA replication reaction. In the parallel experiments we have shown that without DONSON egg extract cannot synthesise nascent DNA. Here we wanted to examine the change of replicating chromatin proteome in absence of DONSON. Interphase Xenopus laevis egg extract was immunodepleted with control nonspecific IgG antibodies beads or DONSON-antibodies. The efficiency of immunodepletion was shown to be over 95%. DNA replication was set up in both IgG- and DONSON-depleted egg extracts and chromatin isolated in the middle of S-phase, when replication forks where abundant in chromatin fraction in IgG-depleted extract.
Project description:Bone marrow stromal cells (BMSCs) are multipotent stem cells that preferentially differentiate into mesenchymal cells. If they can be dedifferentiated into embryonic stem cell-like cells, they will be a highly attractive source for cell therapy. Cell and egg extracts have been used in a few studies to evaluate nuclear reprogramming, but these have not examined cell pluripotency in any detail. In this study, we used a cell reversible permeabilization method to treat BMSC with Xenopus laevis mitotic egg extract. We observed an upregulation of the pluripotent protein Oct3/4 in BMSCs treated by this extract. We also further evaluated transcriptional changes with a focused stem cell oligonucleotide array. A number of genes involved in the Notch or Wnt signaling pathways were upregulated in BMSC exposed to Xenopus egg extract. In conclusion, our microarray data from BMSCs exposure to egg extracts may provide interesting clues regarding factors involved in nuclear reprogramming. Our approach is an alternative method towards dedifferentiation of cells without genetic modification, which is preferable in the clinical situation. Keywords: Cell type comparison Expression profiling was performed on murine bonemarrow stromal cells cultured in-vitro versus stromal cells exposed to Xenopus extract and compared with murine embryonic stem cells
Project description:We analysed dynamics of protein phosphorylation throughout unperturbed early cell cycles in individual Xenopus embryos using high-resolution time-resolved phosphoproteomics, and confirmed cell cycle behaviour in egg extracts.