Development and validation of an in-cell western for quantifying P-glycoprotein expression in human brain microvascular endothelial (hCMEC/D3) cells

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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.

Original languageEnglish
Pages (from-to)2614-2624
Number of pages11
JournalJournal of Pharmaceutical Sciences
Volume106
Issue number9
DOIs
Publication statusPublished - Sep 2017

Keywords

  • ABC transporters
  • Blood-brain barrier
  • Cell culture
  • Drug resistance
  • Efflux pumps
  • Endothelial
  • Membrane transporter
  • P-glycoprotein

Cite this

@article{89eedc50c40c48c1921be68e6c1d3f38,
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.",
keywords = "ABC transporters, Blood-brain barrier, Cell culture, Drug resistance, Efflux pumps, Endothelial, Membrane transporter, P-glycoprotein",
author = "McInerney, {Mitchell P} and Yijun Pan and Short, {Jennifer L.} and Nicolazzo, {Joseph A.}",
year = "2017",
month = "9",
doi = "10.1016/j.xphs.2016.12.017",
language = "English",
volume = "106",
pages = "2614--2624",
journal = "Journal of Pharmaceutical Sciences",
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T1 - Development and validation of an in-cell western for quantifying P-glycoprotein expression in human brain microvascular endothelial (hCMEC/D3) cells

AU - McInerney, Mitchell P

AU - Pan, Yijun

AU - Short, Jennifer L.

AU - Nicolazzo, Joseph A.

PY - 2017/9

Y1 - 2017/9

N2 - 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.

AB - 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.

KW - ABC transporters

KW - Blood-brain barrier

KW - Cell culture

KW - Drug resistance

KW - Efflux pumps

KW - Endothelial

KW - Membrane transporter

KW - P-glycoprotein

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U2 - 10.1016/j.xphs.2016.12.017

DO - 10.1016/j.xphs.2016.12.017

M3 - Article

VL - 106

SP - 2614

EP - 2624

JO - Journal of Pharmaceutical Sciences

JF - Journal of Pharmaceutical Sciences

SN - 0022-3549

IS - 9

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