Incucyte 96 well essen imagelock plate

MDA-MB-231 cells that were transfected with the indicated plasmids (pLKO-EV, sh-CY or sh-ER) were seeded in a IncuCyte 96-well Essen ImageLock plate (4379, Essen BioScience) and scratched using the IncuCyte WoundMaker (Essen BioScience). The scratched cells were washed with PBS and then cultured in the IncuCyte live cell imaging system. Images were acquired every 2 h over a 14–20 h period using a 10 × objective. The relative wound size in each well was analyzed by IncuCyte cell migration software.
Transwell assays were performed in 24-well invasion chambers with an 8.0 µm polyethylene terephthalate membrane (354483, Corning). MDA-MB-231 cells overexpressing USP19-ER-wt or USP19-ER-CS were serum starved overnight and then, seeded into the Transwell inserts, and DMEM supplemented with 10% FBS was added to the lower part of the chamber. The cells inside the chamber were carefully removed by a cotton tip that had been moistened with PBS, and the migrated cells were fixed in 4% paraformaldehyde (PFA, 28908, Thermo Fisher Scientific) for 10 min. These migrated cells were stained with 0.5% crystal violet for 30 min. Five random fields were selected and photographed for each condition, and the number of cells was counted using ImageJ. All the experiments were performed in biological triplicates, and representative results are shown.