Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, VIC, Australia.
Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, VIC, Australia.
J Pharm Sci. 2017 Sep;106(9):2614-2624. doi: 10.1016/j.xphs.2016.12.017. Epub 2017 Jan 5.
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.
建立并优化了一种用于检测人脑微血管内皮细胞（hCMEC/D3）中 P-糖蛋白（P-gp）相对表达的细胞内 Western（ICW）方案，旨在提高比 Western 印迹（WB）更高的通量。为了验证 ICW 方案，用已知的 P-gp 上调剂（10 μM 利福平和 5 μM SR12813）孵育 hCMEC/D3 细胞，并针对 MDR1 靶向 siRNA 进行处理，然后使用 ICW 和 WB 平行测量 P-gp 表达的变化。为了确认检测到的 P-gp 表达与功能之间的关系，在 SR12813 处理后评估了 P-gp 底物罗丹明 123 的摄取。与对照相比，利福平（1.5 倍）和 SR12813（1.9 倍）显著上调了 P-gp 表达，如 ICW 方案所评估的。对相同处理的 WB 分析显示，上调分别为 1.4 倍和 1.5 倍。当通过 ICW 和 WB 评估时，MDR1 siRNA 将 P-gp 丰度降低了 20%和 35%。SR12813 处理将罗丹明 123 的摄取减少了 18%，表明通过 ICW 观察到的 P-gp 表达变化与可比的功能变化相关。WB、罗丹明 123 摄取和 ICW 方案对 P-gp 上调的相关性为在 hCMEC/D3 细胞中定量 P-gp 的新 ICW 方法提供了验证。
