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Label-free proteomic analysis of hepatocyte spheroids fed physiologically relevant levels of palmitic acid and oleic acid to mimic non-alcoholic fatty liver disease.

ABSTRACT: We have grown hepatocyte spheroids for 21 days following an established protocol. We have carried out proteomic analysis of the difference between control and treated (Fatty acids) spheroids during seven days (5 timepoints at day 0, 1, 2, 4 and 7). Samples were digesting using filter aided sample preparation-based digestion and subsequently analysed using label free LC-MS/MS.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Hepatocyte, Liver

DISEASE(S): Non-alcoholic Fatty Liver Disease,Non-alcoholic Fatty Liver

SUBMITTER: Adelina Rogowska-Wrzesinska

LAB HEAD: Adelina Rogowska-Wrzesinska

PROVIDER: PXD029200 | Pride | 2021-11-24

REPOSITORIES: Pride

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Project description:Spheroids, near spherical multicellular aggregates, are one of the most common types of threedimensional (3D) cell cultures. Despite decades of implementation of spheroid technology in various fields of life science and medical research, no minimal information (MI) guidelines are available to cope with heterogeneity and to stimulate transparency. To cope with this unmet need, we assembled an international consortium to develop the MISpheroID knowledgebase (https://www.mispheroid.org) and interrogation revealed heterogeneity and lack of transparency in published spheroid-related experiments. This steered us to empirically evaluate the impact of cell line, culture medium type, spheroid formation method and spheroid size on complementary spheroid metrics (RNA fingerprints, presence of cell death, ATP content, glucose to lactate conversion, secreted protein signatures, circularity, size and cancer therapy response). We measured media-induced transcriptional variation in lung cancer (A549), colorectal cancer (HCT116), ovarium cancer (SKOV3) and glioblastoma (U87MG) spheroids using RNA sequencing (RNA-seq). These spheroids were formed in ultra-low attachment plates and cultured in 6 different media types (DMEM high glucose, DMEM/F12, RPMI1640, DMEM low glucose, EMEM and MEM) for 5 (HCT116) or 7 (A549, SKOV3 and U87MG) days. RNA extraction was performed on 2 spheroids per condition using the miRNeasy micro kit (217084, Qiagen, Hilden, Germany). RNA-sequencing libraries were prepared from purified RNA using the QuantSeq 3' mRNA-Seq Library Prep Kit FWD for Illumina (Lexogen, Vienna, Austria) according to the manufacturer's instructions, using 27.5ng of RNA that was DNase treated using HL-dsDNA (Arcticzymes, TromsØ, Norway). The individual libraries were quantified by qPCR using the KAPA Library Quantification Kit (Roche, Pleasanton, CA, US) and equimolarly pooled. The pool concentration was measured with Qubit and 1.4pM with 1% PhiX was sequenced on a NextSeq 500 (Illumina, San Diego, CA, US) using a high-output 1x75 run. Reads were mapped to the human genome using Tophat and gene expression counts were generated using HTSeq. This data was used to perform Principal Component Analysis (PCA) and Gene Set Enrichment Analysis (GSEA).

			Action	DRS
	Data_from_maxquant_used_for_filtering.xlsx	Xlsx
	Fastafile.fasta	Fasta
	LUM2_03794_LES.raw	Raw
	LUM2_03795_LES.raw	Raw
	LUM2_03796_LES.raw	Raw

Dataset Information

Label-free proteomic analysis of hepatocyte spheroids fed physiologically relevant levels of palmitic acid and oleic acid to mimic non-alcoholic fatty liver disease.

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