RNA-seq of N2 and lin-45(n2018) mutant C.elegans responses to osmotic stress
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ABSTRACT:
ORGANISM(S): Caenorhabditis elegans
PROVIDER: GSE91073 | GEO | 2017/02/06
SECONDARY ACCESSION(S): PRJNA356824
REPOSITORIES: GEO
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Project description:Arabidopsis Col-0 seeds were germinated and grown for two weeks on Arabidopsis thaliana salt media (ATS, control) or ATS media supplemented 50, 75, 100 or 125 mM NaCl that imposes both an ionic and osmotic stress; or ATS media supplemented with iso-osmolar concentrations of sorbitol (100, 150, 200 or 250 mM) that imposes only an osmotic stress. The aim of the study was to identify genes involved in plant growth and adaptation to ionic stress compared to genes involved in growth and adaptation to osmotic stress conditions. To do this we identified lists of genes that are differentially expressed in plants grown in NaCl (A) and lists of genes differentially expressed in plants grown in sorbitol (B). We then compared these lists to find ionic/salt-specific genes that are only expressed in plants grown in NaCl and not in plants grown in sorbitol; and osmotic genes that are expressed both in plants grown in NaCl and in plants grown in sorbitol. Associated publication: Cackett et al. (2022) Salt-specific gene expression reveals elevated auxin levels in Arabidopsis thaliana plants grown under saline conditions, DOI: 10.3389/fpls.2022.804716
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Project description:Human intervertebral disc tissue was obtained from patients (average age 51 yrs) undergoing surgery for lumbar interbody fusion (n=3) or lumbar disc herniation (n=1). Cells were isolated by sequential pronase-collagenase digestion [3]. Cells were passaged twice in monolayer and suspended at a density of 2 x 106 cells/ml in 1.2% alginate (low viscosity, Sigma Chemical, St Louis, MO) dissolved in 150 mM NaCl. Alginate beads were formed by dropwise addition of the alginate from a 22 gauge needle into 102 mM CaCl2, followed by 10 minutes of curing, as described previously [13, 27]. Cell-gel beads were incubated in cell culture media consisting of Ham’s F-12 medium (Gibco BRL, Grand Island, NY), supplemented with 10% FBS (HyClone, Logan, UT), 25 μg/ml ascorbic acid (Sigma, St. Louis, MO), 100 U/ml penicillin, 100 μg/ml streptomycin, and 1 μg/ml Fungizone at 5% CO2 and 37° C. After 24 h, the cell culture medium was removed via pipette and exchanged for one of three osmotically active solutions, representing iso-osmotic, hyper-osmotic and hypo-osmotic media [12, 23, 34]. The iso-osmotic solution consisted of a defined cell culture medium (Ham’s F-12 with supplements as described above; 293 mOsm/kg H2O). The hypo-osmotic solution consisted of the same cell culture media diluted with de-ionized water to a final osmolarity of 250 mOsm/kg H2O. The hyper-osmotic solution consisted of the same cell culture media supplemented with sucrose to a final osmolarity of 450 mOsm/kg H20. The osmolarity of all solution formulations was determined using a freezing-point osmometer (Advanced Laboratory Wide Range 3W2, Advanced Instrument, Needham Heights, MA) as described previously [12]. Cell-alginate beads were cultured for a 4 hour period under one of these conditions, after which the cells were released from alginate in a dissolving buffer (55 mM Na-citrate and 150 mM NaCl), lysed and stored at –80°C. Intervertebral disc (IVD) cells experience a broad range of physical stimuli under physiologic conditions, including alterations in their osmotic environment. In this study, the gene expression profile of human IVD cells was quantified with gene array technology following exposure to varying osmolarity in order to capture the biological responses for a broad set of targets. A total of 42 genes were identified in IVD cells as significantly changed following culture under hyper-osmotic conditions, while a total of 18 genes were identified as significantly changed under hypo-osmotic conditions. Gene expression patterns were verified using RT-PCR. Genes identified in this study include those related to cytoskeleton remodeling and stabilization (ephrin-B2, sarcoglycan beta, IQGAP1), as well as membrane transport (ion transporter SLC21A12, osmolyte tranporter SLC5A3, monocarboxylic acid SLC16A6, and amino acid transporter SLC7A8). An unexpected finding was the differential regulation of the gene for the neurotrophin brain-derived neurotrophic factor by hyper-osmotic stimuli that may be indicative of a physiological response of IVD cells to physical stimuli important in regulating discogenic pain. Keywords = intervertebral disc Keywords = osmotic stimuli Keywords: other
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