Biomedical Engineering Reference
In-Depth Information
3
Methods
3.1 High-Content
Nuclear-Cytoplasmic
Translocation Imaging
Assay
1. Figure
2
illustrates the workfl ow of the assay procedure. The
protocol described in this review uses the U2nesRELOC
system [
13
,
15
] which is an image-based methodology com-
patible with a high-throughput evaluation of small molecule
inhibitors of the nuclear export. The U2nesRELOC is based
on a human osteosarcoma cell line U2OS stably expressing
green fl uorescent protein (GFP)-labeled Rev protein that con-
tains a strong heterologous nuclear export signal (NES). The
fl uorescent signal of untreated U2nesRELOC cells localizes
exclusively to the cytoplasm. Upon treatment with nuclear
export inhibitors, the reporter protein accumulates rapidly in
the cell nucleus.
2. Grow U2nesRELOC cells until they reach 80-90 %
confl uency.
Fig. 2
Schematic workfl ow that illustrates the process of identifi cation and purifi cation of nuclear export inhibi-
tors from natural products
