Dataset Information

Inter-Laboratory Variability in the Madin-Darby Canine Kidney Cell ProteomeInter-Laboratory Variability in the Madin-Darby Canine Kidney Cell Proteome

ABSTRACT: Madin-Darby Canine Kidney (MDCK) cells are widely used to study epithelial cell functionality. Their low endogenous drug transporter protein levels make them an amenable system to investigate trans-epithelial permeation and drug transporter protein activity after their transfection. MDCK cells display diverse phenotypic traits and as such laboratory-to-laboratory variability in drug permeability assessments are observed. Consequently, In Vitro-In Vivo extrapolation (IVIVE) approaches using permeability and/or transporter activity data require calibration. A comprehensive proteomic quantification of eleven filter-grown parental or mock-transfected MDCK monolayers from eight different pharmaceutical laboratories using the Total Protein Approach (TPA) is provided. TPA enabled estimations of key morphometric parameters such as monolayer cellularity and volume. Overall, metabolic liability to xenobiotics is likely to be limited for MDCK cell due to the low expression of required enzymes. SLC16A1 (MCT1) was the highest abundance SLC transporter linked to xenobiotic activity, while ABCC4 (MRP4) was the highest abundant ABC transporter. Our data supports existing findings that Claudin-2 levels may be linked to tight junction modulation, thus impacting trans-epithelial resistance. This unique database provides data on more than 8000 proteins copy number and concentrations, thus allowing an in-depth appraisal of the control monolayers used in each laboratory.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Zea Mays (maize)

TISSUE(S): Liver

SUBMITTER: Jacek Wisniewski

LAB HEAD: Jacek R Wisniewski

PROVIDER: PXD039552 | Pride | 2023-07-20

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	20190528_QX7_JRW_SA_CT_OO43.raw	Raw
	20190528_QX7_JRW_SA_CT_OO46.raw	Raw
	20190528_QX7_JRW_SA_CT_OO50.raw	Raw
	20190528_QX7_JRW_SA_CT_OO56.raw	Raw
	20190528_QX7_JRW_SA_CT_OO58.raw	Raw

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Publications

Interlaboratory Variability in the Madin-Darby Canine Kidney Cell Proteome.

Harwood Matthew D MD Zettl Katharina K Weinheimer Manuel M Pilla-Reddy Venkatesh V Shen Hong H Jacobs Frank F Chu Xiaoyan X Huth Felix F Nakakariya Masanori M Chothe Paresh P PP Neuhoff Sibylle S Wiśniewski Jacek R JR

Molecular pharmaceutics 20230607 7

Madin-Darby canine kidney (MDCK) cells are widely used to study epithelial cell functionality. Their low endogenous drug transporter protein levels make them an amenable system to investigate transepithelial permeation and drug transporter protein activity after their transfection. MDCK cells display diverse phenotypic traits, and as such, laboratory-to-laboratory variability in drug permeability assessments is observed. Consequently, in vitro-in vivo extrapolation (IVIVE) approaches using perme ...[more]

PMID: 37283406

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Project description:Our group is interested in epithelial-to-mesenchymal transition (EMT), in particular, TGF-beta induced EMT. TGF-beta signalling has been shown to be an important factor in the induction of EMT and it has been demonstrated that adding TGF-beta to epithelial cells in culture is a convenient way to study the process of EMT. âIn response to TGF-beta, Smad2 and 3 are activated, and form complexes with Smad4, which then regulate transcription of target genes through interactions with other DNA binding transcription factors. In the induction of EMT, the activated Smads mediate transcriptional regulation through three families of transcription factors, resulting in repression of epithelial marker gene expression and activation of mesenchymal gene expressionâ (Xu J, et al. 2009) Also investigated in this study is the role of H2A.Z in EMT. H2A.Z is an evolutionary conserved and a metazoan essential histone variant of the H2A class. Mice deficient in H2A.Z die during early development but the reason for this is unknown (Faast et al. 2001). Previously, our laboratory showed that the loss of H2A.Z in Xenpous laevis impaired cell movement required for the formation of the mesoderm and neural crest (Ridgway et al. 2004). Given that mesoderm formation is critically dependent upon EMT, we therefore wondered whether H2A.Z might be a chromatin regulator of EMT. We transfected MDCK cells with a lentiviral vector to express a construct encoding an shRNA targeting canine H2A.Z as we wanted to test the hypothesis that H2A.Z is involved in the maintenance of cellular identity and that its loss might trigger de-differentiation. In order to investigate changes in gene expression associated with TGF-beta induced epithelial-to-mesenchymal transition (EMT) we performed paired end RNA-Seq of poly-A selected mRNA in untreated and TGFb-treated MDCK cells. The MDCK cell line has been extensively used as a model system for EMT because they convert fully from the epithelial to the mesenchymal state in response to TGF-beta. Gene expression profiles were also generated from MDCK cells in which H2A.Z was knocked down using shRNA. Please note that ChIP-seq data generated in conjunction to this RNA-seq data set were also deposited at ArrayExpress under accession number E-MTAB-5637 ( https://www.ebi.ac.uk/arrayexpress/experiments/E-MTAB-5637 ).