Figure 1.

shRNA-based selection of positive regulators of cell migration. (A) Overview of the selection procedure. The production and infection of an ubiquitylation-related lentiviral shRNA library are described in Methods. Two weeks after lentiviral infection and selection, MDA-MB-231 cells were seeded onto Boyden chamber inserts and allowed to migrate across the porous membrane at 37 °C for 24 h in order to select cells with a decreased migration phenotype. Migrating cells were removed and non-migrating cells were collected by trypsin treatment from the inserts upper compartment and cultivated for one week. Cells were then reseeded onto Boyden chamber culture inserts for a subsequent round of selection; this procedure was repeated until cells lost 75% of their initial migratory potential. This non-migratory population of cells was then inoculated through tail vein injection into NOD/SCID mice, and primary cultures were generated after two months from lung and brain tissues. shRNAs were retrieved by PCR from the in vitro and in vitro/vivo-selected cells, and then identified by Next Generation Sequencing (NGS). (B) Boyden chamber assay was used every other enrichment cycle to determine the relative percentage of migratory cells and monitor the selection process. (C) shRNAs abundance after the in vivo selection, relative to their abundance in the in vitro-selected non-migratory cells.