Sphingo-1-Identification of Sphingolipid regulated genes in cold stress response. Sphingolipids regulated genes.
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ABSTRACT: rs07-05_sphingolipids-cold - sphingo-1 - The cold choc response seems to be partly triggered by Sphingolipid species. To date no gene response as been associated to sphingolipid signaling pathway in plant. Our aim is to identify among the cold induced genes the ones regulated by sphingolipids and to try to define a sphingolipid pathway specific group of genes. - 7ml of 5 days-old cells suspensions were incubated in presence of different sphingolipid pathway inhibitors, 30 min to 2 hours depending in the coumpound (all were resuspended in DMSO and control were done with DMSO). Then a 30 min cold choc was applied before cells were harvested and frozen in cold nitrogen. RNA were then extracted. FB1 and DMS were from Alexis , Myr from Cayman, TSP from matreya.
Project description:rs07-05_sphingolipids-cold - sphingo-1 - The cold choc response seems to be partly triggered by Sphingolipid species. To date no gene response as been associated to sphingolipid signaling pathway in plant. Our aim is to identify among the cold induced genes the ones regulated by sphingolipids and to try to define a sphingolipid pathway specific group of genes. - 7ml of 5 days-old cells suspensions were incubated in presence of different sphingolipid pathway inhibitors, 30 min to 2 hours depending in the coumpound (all were resuspended in DMSO and control were done with DMSO). Then a 30 min cold choc was applied before cells were harvested and frozen in cold nitrogen. RNA were then extracted. FB1 and DMS were from Alexis , Myr from Cayman, TSP from matreya. 6 dye-swap - treated vs untreated comparison
Project description:Adr1 of Ustilago maydis is a protein kinase that is activated after separation from the regulatory subunit mediated by high cAMP levels. A copy of the gene under the control of the arabinose-inducible crg1 promotor was introduced into the Cbx locus of the wild type strain FB1 creating strain HE140. To monitor the transcriptional changes upon adr1 induction by arabinose, we conducted a time course experiment comparing the wild type transcriptional response before and 75 or 180 min after medium shift to complete medium containing arabinose, as well as the response of the mutant HE140 before and 75 or 180 min after induction of the additional copy of adr1 by arabinose. Keywords: gene induction Strains FB1 (control) and HE140 (a1b1 ipr[pcrg1:adr1]ips) were grown in CM medium containing glucose and were shifted to CM medium containing arabinose and grown for 75 or 180 min. Samples were taken before and after arabinose induction from two (glucose) or three (arabinose) biological replicates each.
Project description:To explored the mechanism by which Myr regulates the biological function of RA FLSs. RNA sequencing was used to identify the differentially expressed transcriptome of Myr-treated RA FLSs compared with the DMSO control.
Project description:Adr1 of Ustilago maydis is a protein kinase that is activated after separation from the regulatory subunit mediated by high cAMP levels. A copy of the gene under the control of the arabinose-inducible crg1 promotor was introduced into the Cbx locus of the wild type strain FB1 creating strain HE140. To monitor the transcriptional changes upon adr1 induction by arabinose, we conducted a time course experiment comparing the wild type transcriptional response before and 75 or 180 min after medium shift to complete medium containing arabinose, as well as the response of the mutant HE140 before and 75 or 180 min after induction of the additional copy of adr1 by arabinose. Keywords: gene induction
Project description:MOLM-13 acute myeloid leukemia cells were treated with 7 µM 5-Azacytidine (Cayman Chemical 11164), or 450 nM CB-5083 (Cayman Chemical 19311), or in combination, or 0.1% DMSO as control. Treatments were conducted for 48 hours.
Project description:Identification of transcriptional changes of Ustilago maydis wild type strain FB1 grown in axenic culture in complete medium containing glucose without or with the addition of 10 µM FeSO4. Keywords: gene induction Strain FB1 (wt) was grown in CM glucose medium in the presence or absence of 10 µM FeSO4.
Project description:1.0 – 1.3 x 10e8 proliferating Jurkat cells at a density of about 1-1.5 x 10e6 cells/mL were employed per sample. Cells were incubated with 0.5mM DMOG (Cayman Chemical Company, 71210) or an equivalent volume of Dimethyl Sulfoxide (DMSO) (vehicle, Merck 1.02950.0500). The DMSO concentration in the medium was 0.023% v/v. Cells were irradiated or not with 254 nm UV light and subjected to eRIC or RIC to determine the RNA-bound proteome. Data correspond to two biologically independent experiments.
Project description:ATP-binding cassette transporters Pdr5 and Yor1 from Saccharomyces cerevisiae control the asymmetric distribution of phospholipids across the plasma membrane as well as serving as ATP-dependent drug efflux pumps. Mutant strains lacking these transporter proteins were found to exhibit very different resistant phenotypes to two inhibitors of sphingolipid biosynthesis that act either late (aureobasidin A:AbA) or early (myriocin: Myr) in the pathway leading to production of these important plasma membrane lipids. These pdr5D yor1 strains were highly AbA resistant but extremely sensitive to Myr. We provide evidence that these phenotypic changes are likely due to modulation of the plasma membrane flippase complexes: Dnf1/Lem3 and Dnf2/Lem3. Flippases act to move phospholipids from the outer to the inner leaflet of the plasma membrane. Genetic analyses indicate that lem3D mutant strains are highly AbA sensitive and Myr resistant. These phenotypes are fully epistatic to those seen in pdr5D yor1 strains. Direct analysis of AbA-induced signaling demonstrated that loss of Pdr5 and Yor1 inhibited the AbA-triggered phosphorylation of the AGC kinase Ypk1 and its substrate Orm1. Microarray experiments found that a pdr5D yor1 strain induced a Pdr1-dependent induction of the entire Pdr regulon. Our data support the view that Pdr5/Yor1 negatively regulate flippase function and activity of the nuclear Pdr1 transcription factor. Together, these data argue that the interaction of the ABC transporters Pdr5 and Yor1 with the Lem3-dependent flippases regulate permeability of AbA via control of plasma membrane protein function as seen for the high-affinity tryptophan permease Tat2. the transcriptome of pdr5Dyor1D double mutants was compared either to wild type cells or to pdr5Dyor1Dpdr1D triple mutant.
Project description:Identification of transcriptional changes of Ustilago maydis wild type strain FB1 grown in axenic culture in complete medium containing glucose without or with the addition of 10 µM FeSO4. Keywords: gene induction