Project description:Lipid droplet (LD) function is regulated by a complement of integral and peripheral proteins that associate with the LD phospholipid monolayer. Defining the composition of the LD proteome has remained a challenge due to the presence of contaminating proteins in LD-enriched buoyant fractions. To overcome this limitation, we developed a proximity labeling strategy that exploits LD-targeted APEX2 to biotinylate LD proteins in living cells. Application of this approach to U2OS and Huh7 cells identified the vast majority of previously validated LD proteins, excluded common contaminating proteins, and revealed new LD proteins.
Project description:One of the key functions of the mammalian liver is lipid metabolism. During fasting, lipid storage in the liver increases in order to reserve and provide energy for cellular functions. Upon re-feeding, this reserve of lipids is rapidly depleted; this change is visible, as the organelles responsible for lipid storage – lipid droplets (LDs) – drastically decrease in size following re-feeding. Little is known regarding LD proteome, or how it changes during the fasting/re-feeding transition. Our study investigated the hepatic LD proteome and how it changes between fasting and re-feeding conditions. For this purpose, LDs were isolated from 4 month-old C57BL/6 mice after a 24 hour fasting period, or a 24 hour fasting period followed by 6 hours of re-feeding. Proteins isolated from these LDs were subject to SDS-PAGE followed by in-gel trypsinization and LC-MS/MS. We identified a combined total of 941 proteins on hepatic LDs, of which 817 had quantifiable extracted ion chromatograms in at least 2 samples (n=6 total) and were not deemed contaminants. 777 of the 817 proteins were observed in both energetic states, with 33 being uniquely observed in fasted LDs, and 7 being uniquely observed in re-fed LDs.
Project description:Metarhizium robertsii is one of the model fungus species widely used studying insect-fungus interactions. Our previous studies have shown the accumulation of lipid droplets (LDs) play an essential role in generation of cellular turgor pressure to assist fungal penetration of insect cuticles, and autophagy-related proteins are connected with LD biogenesis and cellular accumulation in M. robertsii. However, full proteomes of LDs are unclear in terms of the species of proteins involved in lipid biosynthesis and degradation. We performed experiments by growing the fungi in different nutrient conditions, isolated the LDs and extracted the LD proteins from the cultures after being grown in a nutrient-rich medium (i.e. Sabouraud dextrose broth, SDB), a minimum medium without nitrogen source (MM-N) and MM-N supplemented with oleate. The protein samples were then subject to LC-MS/MS proteome profilings for identifying and postulating the proteins/pathways involved in lipid metabolisms.
Project description:Genome expression analysis between the yeast wine strain L-846 (diploid heterozygous) and spore derived from it (diploid homozygous). Slides contained cDNA clones spotted in duplicate. A repeated dye-swap design was used.
Project description:This experiment was designed to study the effects of lipid culture on INS-1 derived glucose-responsive 832/13 cells. After 48-hr culture with lipids, a dramatic decrease in glucose-responsiveness is observed. In order to detect some earlier changes, RNA was also collected at earlier time points (12 hr and 24 hr). To compensate for any changes associated with cell density, control cells (1%BSA culture) were included for each time point. RNA was prepared from INS-1 cells in Dr. Chris Newgard's lab, quantified and sent frozen in water. Six biological replicates were sent for the following conditions: (i) 1%BSA (12, 24, and 48 hours) (ii) 1mM-Oleate/Palmitate (12, 24, and 48 hours).
Project description:The aim of this experiment is to compare nucleases used in ribosome profiling using Drosophila melanogaster Kc167 cells and human U2OS osteosarcoma cells. Ribosome profiling was performed from nuclease digested samples using sucrose cushion centrifugation separated ribosomes.
Project description:Ribosome profiling (Ribo-Seq) (maps positions of translating ribosomes on the transcriptome) analysis of human (RD) cells infected with enterovirus strains EV7, EV71, and PV1.
Project description:Ribosome profiling is a technique that permits genome-wide, quantitative analysis of translation and has found broad application in recent years. A key step is the depletion of ribosomal RNA that allows sufficient depth of sequencing of the mRNA undergoing ribosomal translation. These data are designed to compare four strategies for ribosomal depletion; a novel strategy based on duplex-specific nuclease using either one or two cycles, the antisense oligonucleotides described in Ingola et al (2012) and the commercially available RiboZero kit (with a no treatment control).
Project description:Ribosome profiling data from U2OS, HeLa and Kc167 cells under various lysis conditions and using immunoprecipitation to purifiy ribosome associated footprints. Two human cell lines (U2OS and HeLa cells) and a Drosophila melanogaster cell line (Kc167) are used to see if the 3'UTR reads are identified in each cell type. Immunoprecipitation of ribosomes is used to analyse if 3'UTR reads derive from ribosomes (are found with ribosome immunoprecipitates) and to which extent the lysis conditions contribute to the identification of the 3'UTR reads.