Project description:Study of the emergence of the rare 2C like cell population upon Retinoic Acid treatment. Transcriptionally characterise the different cell populations emerging at different timepoints upon Retinoic Acid treatment and identify genes driving cell fate decisions.
Project description:CIARA (Cluster Independent Algorithm for the identification of RAre cell types) is tested on mouse embryonic stem cell treated for 24h with Retinoic Acid.
Project description:Many new alternative splice forms have been detected at the transcript level using next generation sequencing (NGS) methods, especially RNA-Seq, but it is not known how many of these transcripts are being translated. Leveraging the unprecedented capabilities of NGS, we collected RNA-Seq and proteomics data from the same cell population (Jurkat cells) and created a bioinformatics pipeline that builds customized databases for the discovery of novel splice-junction peptides. Results: Eighty million paired-end Illumina reads and ~500,000 tandem mass spectra were used to identify 12,873 transcripts (19,320 including isoforms) and 6,810 proteins. We developed a bioinformatics workflow to retrieve high-confidence, novel splice junction sequences from the RNA data, translate these sequences into the analogous polypeptide sequence, and create a customized splice junction database for MS searching. Jurkat T-cell mRNA was analyzed on an Illumina HiSeq2000. ~80 million paired end reads (2x200bp, ~350bp lengths) were collected.
Project description:This study demonstrates cellular and molecular mechanisms regulating the generation and function of basal radial glia (bRG), a neural stem cell population that was shown to be enriched in the developing human brain and was involved in the formation of cortical expansion. Here, we studied the role of LGALS3BP, a secreted protein whose RNA expression is enriched in bRGs. Based on a combination of three model systems i) in vitro human cerebral organoids, ii) ex vivo embryonic human fetal tissue and iii) in vivo embryonic mouse cortex, and a wide variety of techniques including single-cell RNA-sequencing, proteomics, and immunostaining, we characterized the role of human LGALS3BP mutations.
Project description:In addition to driving tumorigenesis, oncogenes can create metabolic vulnerabilities in cancer cells. Here, we tested how the oncogenes AKT and MYC affect the ability to shift between respiration and glycolysis. Using immortalized mammary epithelial cellsMCF10A, we discovered constitutively active AKT but not MYC induced cell death in galactose culture, where cells must rely on oxidative phosphorylation for energy generation. However, the negative effects of AKT were short-lived, and AKT-expressing cells recommenced growth after ~15 days in galactose culture. To identify the mechanisms regulating AKT-mediated cell death, we first used metabolomics and found that AKT cells dying in galactose culture exhibited upregulated glutathione metabolism. Next, using shotgun proteomics, we discovered AKT cells dying in galactose upregulated proteins related to nonsense-mediated mRNA decay (NMD), a known response to oxidative stress. We therefore measured levels of reactive oxygen species (ROS) and discovered galactose culture induced ROS only in cells expressing AKT. Additionally, we found thatdiscovered the ROS scavenger catalase rescued AKT-expressing cells from galactose culture-induced cell death. We then demonstrated that breast cancer cell lines with constitutively active AKT signaling also exhibited cell death in galactose culture and rescue by catalase. Together, our results demonstrate that AKT but not MYC induces a metabolic vulnerability in cancer cells, namely the that restricted flexibility to use oxidative phosphorylation.
Project description:Cell lines used to determine the growth of cell lines over time were used for transcriptomic profiling to build a model that predicts growth rate from transcriptome of cells. To establish robust transcriptional signatures of dying cells, we performed single-well cell sorting followed by Smart-seq3xpress. 4T1, A549, MDA-MB-231 clone A, L and S were grown in cell culture medium. Adherent MDA-MB-231 clone L were grown with 1uM of Paclitaxel, a concentration we have previously shown to slow proliferation while retaining a viability compatible with the experiment27. The cells in culture were detached with Trypsin treatment for 3-5 minutes at 37 degrees C and the reaction was quenched by adding back the cells supernatant to conserve dead cells. The suspension was washed twice in PBS, 4 minutes at 400g. The cells were then labelled using the live/dead stain BOBO3 and apoptosis marker AnnexinV-APC following manufacturer's instruction. We isolated 3 cell populations on the basis of the BOBO3 and Annexin-V flow cytometry markers using an Aria3 flow cytometry with a 100um nozzle and low pressure settings. Live cells were gated as BOBO3- AnnexinV-, dying (early apoptotic) cells as BOBO3- AnexinV+ and dead cells (necrosis or late apoptosis) as BOBO3+ AnexinV+ (Fig. S2a-b). Individual cells were isolated in 384 well pcr-plates. After 10s centrifugation at 1000g the plates were sealed and stored at -80*C until processed using Smart-seq3xpress protocol30. All the reagents and bioinformatic pipeline related to Smart-seq3xpress are available online31.