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Cross-talks between Metabolic and Translational Controls during Beige Adipocyte Differentiation

ABSTRACT: Measurement of protein levels upon beige adipocyte differentiation. Adipocyte precursor cells were isolated from inguinal white adipose tissue, and induction of beige adipocyte differentiation was performed. Non-induced and differentiated samples for 4 or 8 days were used for sample preparation. For each condition, triplicates were constructed. Peptide fragmentation was performed for 9 samples, and then an equal volume of each sample was pooled to generate Ref 1 and 2. Next, a total of 11 samples were labeled with 11 different TMT reagents. Labeled samples were pooled into one. Then, 11 fractions were acquired from the HPLC fraction system, and LS-MS/MS of each fraction was performed.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Mus Musculus (ncbitaxon:10090)

SUBMITTER: Mihye Lee Jun Cho

PROVIDER: MSV000093396 | MassIVE | Mon Nov 13 22:09:00 GMT 2023

SECONDARY ACCESSION(S): PXD046925

REPOSITORIES: MassIVE

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Project description:Lycopersicon esculentum cv. Moneymaker tomato plants were grown for 4 weeks in greenhouse conditions (16h/8h light/darkness, 27 °C). L. esculentum was randomly sorted into two groups of 16 plants each and a third reference group of 6 plants. All plants were contained individually in mesh bags. One group of 16 plants was infested with 25 female adult tomato psyllids that were 2-3 days old per plant. The other two groups of 16 and 6 plants were not infested (healthy control, and reference pool). After 3 days, the adults were removed from the infested plants to allow for egg eclosion and eventually nymphal development. In this study, leaves infested and healthy plants were harvested for RNA extraction at 0, 3, 11, and 17 days after infestation with tomato psyllids. The reference pool was harvested on day 0. Tissue (all-leaflets) from at least three plants per group was collected, flash frozen in liquid nitrogen and pooled for each time point (0, 3, 11 and 17 days after infestation) to represent treatments for before feeding (0 d), adult feeding and egg deposition (3 d), 1st and 2nd instar feeding (11 d) and 3rd to 5th instar feeding (17 d ), respectively. The whole experiment was repeated three times (three biological replicates). Plant tissues were ground using a cold mortar and pestle. Total tomato leaf RNA was extracted using the hot-phenol protocol. The RNA was precipitated, pooled, cleaned with a kit and stored at –80C. Two populations of single-stranded cDNAs will be generated from the aliquots and labeled with the cyanine Cy3 and Cy5 fluorophores. RNA isolated from the reference pool (0 d) will be pooled from each replicate and will be labeled with Cy5; this is the reference sample for each hybridization. RNA isolated from leaves infested with tomato psyllids and healthy uninfested plants from each time point will be query samples labeled with Cy3. The two samples of labeled cDNA will be simultaneously hybridized to the same microarray. Keywords: Reference design

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Cross-talks between Metabolic and Translational Controls during Beige Adipocyte Differentiation

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