In-cell Western™ detection of organic cation transporters in bronchial epithelial cell layers cultured at an air–liquid interface on Transwell® inserts

• Published in: Journal of pharmacological and toxicological methods Vol. 68; no. 2; pp. 184 - 189
• Main Authors: Mukherjee, Manali, Latif, M.L., Pritchard, D.I., Bosquillon, C.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.09.2013
• Subjects: Blotting, Western - methods; Bronchi - cytology; Bronchi - metabolism; Calu-3 cell line; Cell Line; Drug absorption; Drug transporters; Epithelial Cells - metabolism; Epithelium; Fenofibrate - pharmacology; Glyceraldehyde-3-Phosphate Dehydrogenases - immunology; Glyceraldehyde-3-Phosphate Dehydrogenases - metabolism; Humans; In vitro models; Methods; Nitroprusside - pharmacology; Organic Cation Transport Proteins - genetics; Organic Cation Transport Proteins - immunology; Organic Cation Transport Proteins - metabolism; Protein quantification; Reproducibility of Results; Thiazolidinediones - pharmacology; Transwell; OCT; RT; In vitro models; Drug absorption; PFA; WB; FACS; BSA; SNP; PPAR; Calu-3 cell line; FBS; NHBE; qPCR; w.r.t; IR; Epithelium; Protein quantification; Transwell; Drug transporters; NIR; TEER; SEM; ICW; GAPDH; ALI; Methods; Western blot; room temperature; with respect to; trans-epithelial electrical resistance; fluorescence-activated cell sorting; near infra-red; foetal bovine serum; sodium nitroprusside; organic cation transporter; normal human bronchial epithelial; glyceraldehyde-3-phosphate dehydrogenase; paraformaldehyde; air liquid interface; peroxisome proliferator activated receptor; quantitative polymerase chain reaction; standard error of the mean; bovine serum albumin; in-cell Western; infra red
• ISSN: 1056-8719; 1873-488X
• DOI: 10.1016/j.vascn.2013.05.007

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. 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. 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. 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.