title: In-cell Western™ detection of organic cation transporters in bronchial epithelial cell layers cultured at an air-liquid interface on Transwell(®) inserts.
abstract: BACKGROUND
  Organic cation transporters (OCT) have been shown to mediate the transport of inhaled drugs in bronchial epithelial cells and might have important physiological functions in the airway epithelium. However, a quantitative method to evaluate OCT protein expression in physiologically relevant airway epithelial cell culture models is currently lacking. In-cell Western™ (ICW) techniques might fill that gap but to date, have only been performed on cells grown on 96 or 384-well microplates.
  
  
  METHODS
  An ICW assay was designed for measuring levels of the different OCT subtypes in intact layers of the human bronchial epithelial Calu-3 cell line cultured at an air-liquid interface on Transwell(®) inserts. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the internal standard for normalisation of cell number between the layers. The protocol was subsequently validated by exposing cell layers to compounds known to cause variations in OCT expression.
  
  
  RESULTS
  Antibody signals above the background fluorescence were detected for OCT1, OCT3, OCTN1 and OCTN2 but not for OCT2 in 21day old Calu-3 layers, in agreement with previous studies which had reported OCT2 was absent in the Calu-3 cell line. Furthermore, increases in the fluorescence signal associated with OCT1, OCTN1 and OCTN2 were obtained following treatment of the layers with, respectively, the nitric oxide inducer sodium nitroprusside, the peroxisome proliferator activated receptor α (PPARα) agonist fenofibrate or the PPARγ agonist rosiglitazone, confirming the reliability of the ICW method developed. However, a suitable positive control for OCT3 could not be identified.
  
  
  CONCLUSIONS
  This novel ICW assay can be exploited to quantify basal OCT protein expression as well as changes in transporter levels following external stimuli in various in vitro models. It can also be easily adapted to probe any protein in epithelial layers maintained on permeable filters.
authors.affiliation: Division of Drug Delivery and Tissue Engineering, School of Pharmacy, University of Nottingham, University Park, Nottingham NG7 2RD, United Kingdom.
authors.forename: Manali
authors.initials: M
authors.lastname: Mukherjee
authors.affiliation: School of Biomedical Sciences, Queens Medical Centre, University of Nottingham, Nottingham NG7 2UH, United Kingdom.
authors.forename: M L
authors.initials: ML
authors.lastname: Latif
authors.affiliation: Immune Modulation Research Group, School of Pharmacy, University of Nottingham, University Park, Nottingham NG7 2RD, United Kingdom.
authors.forename: D I
authors.initials: DI
authors.lastname: Pritchard
authors.affiliation: Division of Drug Delivery and Tissue Engineering, School of Pharmacy, University of Nottingham, University Park, Nottingham NG7 2RD, United Kingdom. Electronic address: cynthia.bosquillon@nottingham.ac.uk.
authors.forename: C
authors.initials: C
authors.lastname: Bosquillon
mesh_terms: D015153:Blotting, Western / Q000379:methods*; D001980:Bronchi / Q000166:cytology / Q000378:metabolism*; D002460:Cell Line; D004847:Epithelial Cells / Q000378:metabolism*; D011345:Fenofibrate / Q000494:pharmacology; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000276:immunology / Q000378:metabolism; D006801:Humans; D009599:Nitroprusside / Q000494:pharmacology; D027701:Organic Cation Transport Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D015203:Reproducibility of Results; D000077154:Rosiglitazone; D045162:Thiazolidinediones / Q000494:pharmacology
chemical_list: D027701:Organic Cation Transport Proteins; D045162:Thiazolidinediones; D000077154:Rosiglitazone; D009599:Nitroprusside; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D011345:Fenofibrate
keywords: ALI; BSA; Calu-3 cell line; Drug absorption; Drug transporters; Epithelium; FACS; FBS; GAPDH; ICW; IR; In vitro models; Methods; NHBE; NIR; OCT; PFA; PPAR; Protein quantification; RT; SEM; SNP; TEER; Transwell; WB; Western blot; air liquid interface; bovine serum albumin; fluorescence-activated cell sorting; foetal bovine serum; glyceraldehyde-3-phosphate dehydrogenase; in-cell Western™; infra red; near infra-red; normal human bronchial epithelial; organic cation transporter; paraformaldehyde; peroxisome proliferator activated receptor; qPCR; quantitative polymerase chain reaction; room temperature; sodium nitroprusside; standard error of the mean; trans-epithelial electrical resistance; w.r.t.; with respect to