In cell investigator software v1

The cell lines were seeded at a density of 10,000 cells/well in 48-well plates (Thermo Scientific Nunclon^TM Delta Surface-150687). During immunocytochemistry, the growth medium was discarded, and cells were washed thrice gently with Dulbecco’s modified PBS (DPBS; pH 7.5). Briefly, cells were fixed in 4% paraformaldehyde ± 0.1% Triton X-100 (depending on the desired permeabilization conditions), rinsed with DPBS, and incubated with primary antibodies at 4 °C overnight. After rinsing again in DPBS, cells were incubated with an appropriate fluorescence-conjugated secondary antibody (Supplementary Table S2) and with diamidino phenyl indole (DAPI) as a nuclear stain (diluted to a final concentration of 1 µg/mL) for 2 h at room temperature in the dark with gentle rotary shaking. The plates were then washed thrice with DPBS and images were captured on a high-content imaging platform (Cytell Cell Imaging System (GE Healthcare) or IN Cell Analyser 6000 (GE Healthcare, Buckinghamshire, UK), as indicated), with approximately 6–9 fields of view taken per well. Images were further analysed and quantified using the IN Cell Investigator software v1.0 (GE Healthcare).

The EpCAM sorted subpopulation, parental PMC42-LA cells and the single cell-derived clones were seeded at a density of 10,000 cells/well in 48-well plates (Thermo Scientific Nunclon^TM Delta Surface-150687). During immunocytochemistry, the growth medium was discarded, and cells were washed thrice gently with Dulbecco’s modified phosphate-buffered saline (DPBS; pH 7.5). Briefly, cells were fixed in 4% paraformaldehyde ± 0.1% Triton X-100 (depending on the desired permeabilization conditions), rinsed with DPBS, and incubated with the designated primary antibodies at 4 °C overnight. After rinsing in DPBS, cells were incubated for 2 h at room temperature in the dark on a gentle rotary shaker with appropriate fluorescence-conjugated secondary antibody (Supplementary Table S2) and with diamidino phenyl indole (DAPI) as a nuclear stain (diluted to a final concentration of 1 µg/mL). The plates were then washed thrice with DPBS and images captured on a high-content imaging platform (Cytell Cell Imaging System (GE Healthcare, Buckinghamshire, UK), IN Cell Analyzer 6000 (GE Healthcare, Buckinghamshire, UK) or PerkinElmer Operetta® (PerkinElmer, Waltham, MA, USA) as indicated) with approximately 9 fields of view taken per well. Images were analyzed and merged using the respective software; IN Cell Investigator software v1.0 (GE Healthcare) or Harmony® v4.8 (PerkinElmer).