title: Development and Validation of an In-Cell Western for Quantifying P-Glycoprotein Expression in Human Brain Microvascular Endothelial (hCMEC/D3) Cells.
abstract: An in-cell western (ICW) protocol detecting the relative expression of P-glycoprotein (P-gp) in human cerebro-microvascular endothelial cells (hCMEC/D3) was developed and optimized, with the intention of improving throughput relative to western blotting (WB). For validation of the ICW protocol, hCMEC/D3 cells were incubated with known P-gp upregulators (10 μM rifampicin and 5 μM SR12813) and treated with siRNA targeted against MDR1, before measuring changes in P-gp expression, using both ICW and WB in parallel. To confirm a relationship between the detected P-gp expression and function, the uptake of the P-gp substrate rhodamine-123 was assessed following SR12813 treatment. Rifampicin and SR12813 significantly upregulated P-gp expression (1.5-fold and 1.9-fold, respectively) compared to control, as assessed by the ICW protocol. WB analysis of the same treatments revealed 1.4-fold and 1.5-fold upregulations. MDR1 siRNA reduced P-gp abundance by 20% and 35% when assessed by ICW and WB, respectively. SR12813 treatment reduced rhodamine-123 uptake by 18%, indicating that the observed changes in P-gp expression by ICW were associated with comparable functional changes. The correlation of P-gp upregulation by WB, rhodamine-123 uptake, and the ICW protocol provide validation of a new ICW method as an alternative method for quantification of P-gp in hCMEC/D3 cells.
authors.affiliation: Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, VIC, Australia.
authors.forename: Mitchell P
authors.initials: MP
authors.lastname: McInerney
authors.affiliation: Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, VIC, Australia.
authors.forename: Yijun
authors.initials: Y
authors.lastname: Pan
authors.affiliation: Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, VIC, Australia.
authors.forename: Jennifer L
authors.initials: JL
authors.lastname: Short
authors.affiliation: Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, VIC, Australia. Electronic address: joseph.nicolazzo@monash.edu.
authors.forename: Joseph A
authors.initials: JA
authors.lastname: Nicolazzo
mesh_terms: D018435:ATP Binding Cassette Transporter, Subfamily B / Q000378:metabolism*; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000235:genetics / Q000378:metabolism*; D001692:Biological Transport; D001812:Blood-Brain Barrier / Q000378:metabolism; D015153:Blotting, Western / Q000379:methods*; D001921:Brain / Q000098:blood supply / Q000378:metabolism*; D002138:Calibration; D002560:Cerebrovascular Circulation; D004351:Drug Resistance; D042783:Endothelial Cells / Q000378:metabolism; D004730:Endothelium, Vascular / Q000166:cytology* / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry; D015870:Gene Expression; D006801:Humans; D034741:RNA, Small Interfering / Q000235:genetics; D012293:Rifampin / Q000494:pharmacology; D015854:Up-Regulation
chemical_list: D018435:ATP Binding Cassette Transporter, Subfamily B; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D005456:Fluorescent Dyes; D034741:RNA, Small Interfering; D012293:Rifampin
keywords: ABC transporters; P-glycoprotein; blood-brain barrier; cell culture; drug resistance; efflux pumps; endothelial; membrane transporter