Extended Data Fig. 4: High-resolution imaging of competition-induced Loser cell death.

a, High-resolution live-cell imaging experiment (28 h) showing cell competition in MCF-7 hFWE^KO cells expressing hFWE1–IRES–RFP and hFWE2–IRES–GFP isoforms. The co-culture results show elimination of cells carrying the Lose isoform (hFWE1–IRES–GFP); n = 3 biologically independent experiments with similar results. b, The results of live-cell imaging were confirmed using annexin-V staining and flow-cytometry-based imaging of GFP⁺ and RFP⁺ cells for each co-culture combination. Cells were sorted following 24 h of co-culture, and the percentage of apoptotic cells is displayed. The flow-based imaging of these cells is also presented and shows no GFP⁺ signal in RFP-sorted cells or RFP⁺ signal in GFP-sorted cells. The annexin-V⁺ signal is shown in purple. For example, co-culture of cells expressing hFWE1–IRES–GFP with cells expressing hFWE2–IRES–RFP results in apoptosis of hFWE1–IRES–GFP cells, as indicated by the annexin-V⁺ signal. Together, results from live-cell imaging and flow cytometry demonstrate that cells expressing hFWE^Lose isoforms (hFWE1 or hFWE3) undergo apoptosis when in competition with cells expressing hFWE^Win isoforms (hFWE2 or hFWE4) (n = 3 biologically independent experiments, P values shown, two-tailed t-test, was performed for P-value calculations, mean ± s.d.).

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