Fig. 2

Leader cells do not represent a fixed lineage. A LC-T2A-GFP lines were single cell sorted based on LC^GFP+/− status into a single well of a half area 96-well plate and imaged daily at 4X magnification under bright field, for GFP status using the Cytation™ 3 Multimode Imager, scale represents 1000 μm. B LC-T2A-GFP lines were labelled with the pro-dye CellTrace™ Blue (CTB) and routinely imaged for LC^GFP+ and CTB status over a seven-day period using the Cytation™ 3 Multimode Imager. Data are representative of duplicate experiments with 4 imaging areas per triplicate well and are presented as the mean. Representative data from COV362.4-T2A-GFP cells are shown where n = 3 from two experimental replicates. C Flow cytometric analysis of Ki67 surface expression in LC^+/− populations. Cells were seeded at 300,000 cells/well in a 6-well plate, incubated for 18 h, collected and stained for Ki67 using a Ki67-BV786 antibody. GFP (indicative of KRT14 expression) and BV786 fluorescence was acquired using the BD LSRFortessa™ X-20 and data was analysed using FlowJo software (v10.5.0). Analysis was performed using an unpaired non-parametric Mann-Whitney U test to determine statistical significance between groups; n = 2–4/cell line; one representative cell line (SKOV3) is shown (error bars indicate n = 2 replicates); ns = not significant