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Isolation of RBP47 interactome

ABSTRACT: Stress granules are dynamic structures, in which components belonging to the shell structure can exchange rapidly while components in the core layer may be less dynamic. The last group of proteins has been suggested to act as a scaffold to which other SG components are recruited. In a previous study, we observed that TSN does not exchange between the cytoplasm and foci upon a fluorescence recovery after photobleaching (FRAP) analysis, suggesting a role as a scaffold protein. Deletion of scaffold-like molecules has been suggest to have a high impact in membraneles organelles composition. Accordingly, and to better understand the role of TSN as a platform for SG components in plants, we characterized the SG proteome by isolation of GFP-tagged RBP47 protein from WT and double tsn1 tsn2 knock-out expressing plants.

ORGANISM(S): Arabidopsis Thaliana (mouse-ear Cress)

SUBMITTER: Emilio Gutierrez-Beltran

PROVIDER: PXD024962 | JPOST Repository | Fri Jun 11 00:00:00 BST 2021

REPOSITORIES: jPOST

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Project description:Autologous chondrocyte transplantation (ACT) is a routine technique to regenerate focal cartilage lesions. However, patients with osteoarthritis (OA) are lacking an appropriate long-lasting treatment alternative, partly since it is not known if chondrocytes from OA patients have the same chondrogenic differentiation potential as chondrocytes from donors not affected by OA. Articular chondrocytes from patients with OA undergoing total knee replacement (Mankin Score >3, AhlbÃ¤ck Score >2) and from patients undergoing ACT, here referred to as normal donors (ND), were isolated applying protocols used for ACT. Their chondrogenic differentiation potential was evaluated both in high-density pellet and scaffold (Hyaff-11) cultures by histological proteoglycan assessment (Bern Score) and immunohistochemistry for collagen types I and II. Chondrocytes cultured in monolayer and scaffolds were subjected to gene expression profiling using genome-wide oligonucleotide microarrays. Expression data were verified by using quantitative RT-PCR. Chondrocytes from ND and OA donors demonstrated accumulation of comparable amounts of cartilage matrix components, including sulphated proteoglycans and collagen types I and II. The mRNA expression of cartilage markers (COL2A1, COMP, aggrecan, CRTL1, SOX9) and genes involved in matrix synthesis (biglycan, COL9A2, COL11A1, TIMP4, CILP2) was highly induced in 3D cultures of chondrocytes from both donor groups. Genes associated with hypertrophic or OA cartilage (COL10A1, RUNX2, periostin, ALP, PTHR1, MMP13, COL1A1, COL3A1) were not significantly regulated between the two groups of donors. The expression of 661 genes, including COMP, FN1, and SOX9, were differentially regulated between OA and ND chondrocytes cultured in monolayer. During scaffold culture, the differences diminished between the OA and ND chondrocytes, and only 184 genes were differentially regulated. Only few genes were differentially expressed between OA and ND chondrocytes in Hyaff-11 culture. The risk of differentiation into hypertrophic cartilage does not seem to be increased for OA chondrocytes. Our findings suggest that the chondrogenic capacity is not significantly affected by OA and OA chondrocytes fulfill the requirements for matrix-associated ACT. Experiment Overall Design: Gene expression profiles of monolayer cultures (ML; passage 2) and Hyaff-11 scaffold cultures (3D; 14 days in vitro) of chondrocytes from 3 normal donors (ND; underwent ACT treatment) and 3 donors suffering from Osteoarthritis (OA; underwent knee replacement surgery) were determined. Comparative analyses between 3D and ML cultures (3D vs. ML) were performed to assess differentiation capacity of ND and OA chondrocytes. Furthermore, OA-related differences were determined comparing OA and ND monolayers as well as scaffold cultures (each OA vs. ND).

Project description:The aim of this study was to determine the changes in gene expression of rice root tips when they came in to contact with a hard layer (60% wax layer). Three categories of root tips were sampled; tips before the hard layer, tips that had come into contact with the hard layer and root tips which had buckled after coming into contact with the hard layer. Two genotypes (Azucena and Bala) that vary in there ability to penetrate a hard layer were selected for a genotype comparison of gene expression at the hard layer. Experiment Overall Design: A chamber with the internal dimensions of 10 x 5 x 25cm consisting of a glass back, Perspex front and aluminium spacers with an open bottom and top was set at an angle of 15o in a large sand filled tray and filled up to 19.5 cm with dry sand. The sand was watered with nutrient solution, the sand was levelled and then a 5mm thick 60% wax layer (prepared using a ratio of 3:2 (w/w) of pastillated paraffin wax / white soft wax) was poured on the sand. Once the wax layer had set, two small holes were placed at the back of the wax layer to allow water drainage and water exchange via capillary action. The chamber was then filled up with more dry sand, and then watered using nutrient solution. Nutrient solution was then added to the large tray which the chamber had been placed; this was regularly topped up and allowed the watering of the chamber by capillary action. Light was eliminated from the chambers using white (exterior face)/black (interior face) plastic sheeting. Experiment Overall Design: Azucena and Bala seeds were surface sterilized with 1% (v/w) sodium hypochlorate for 5 minutes, then rinsed thoroughly three times with water and then germinated on moist tissue paper for 48 hours at 37oC. A single germinated seed was then sown 1 cm below the surface of the sand in each chamber. Experiment Overall Design: The plants were grown in a controlled environment room with a 12 hour day and night, with a day temperature of 30oC and a night temperature of 24oC with 300 Âµmol m-2 s-1 PAR. After 24 days the chamber was dismantled and the root tips of 5 mm length were harvested from Azucena plants for subsequent RNA extractions from the three categories as follows; a) roots which were between 5-10 mm above the wax layer; b) roots at the wax layer; c) roots which had buckled at the wax layer and grown 7-10 mm after buckling. From Bala plants, only root tips at the layer were harvested. Experiment Overall Design: Each condition was indsependently replicated 3 times.

			Action	DRS
	A_ECL00924.mgf	Mgf
	A_ECL00924.raw	Raw
	B_ECL01367-2.mgf	Mgf
	B_ECL01367-2.raw	Raw
	C_ECL01368-2.mgf	Mgf

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Isolation of RBP47 interactome

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