Figure 1.

Standard cell competition assay and high‐throughput chemical compound screening. (a) Schematic diagram showing both the standard cell competition assay and the protocol used to screen for chemical compounds promoting the apical extrusion of YAP (5SA) cells during cell competition. In both cases, Dox‐inducible YAP (5SA) cells were labeled with CMTPX (red fluorescent dye), mixed 1:50 with unlabeled normal MDCK cells and incubated in cocultures for 24 hr. For standard cell competition assays, cocultures were incubated for another 24 hr with Dox before fixation. Labeled, apically extruded cells were detected by phalloidin staining and confocal microscopy. For high‐throughput chemical screening, cocultures were incubated for 72 hr with Dox with/without compounds before fixation. Aggregated cells were examined by phase‐contrast microscopy. (b) Representative images of cell aggregation in the high‐throughput screening protocol where CMTPX‐labeled YAP (5SA) cells were mixed 1:50 with unlabeled normal MDCK cells and treated (as indicated) for 72 hr with Dox plus LY294002 (LY, PI3K inhibitor), Rapamycin (Rap, mTOR inhibitor) or PF‐4708671 (PF, S6K inhibitor), starting at 24 hr postseeding. Controls were cocultures incubated for 72 hr without Dox or with Dox plus DMSO. “Bright,” phase‐contrast image. “CMTPX,” red fluorescence image. (c) Quantification of numbers of cell aggregates in the cocultures in (b). Data are the mean +SD (n = 3/group) of three independent experiments