Extended Data Fig. 3: Live-cell imaging of co-culture assay using MCF-7 hFWEKO cells transduced with hFWE isoforms.
a, Results from 24-h live-cell imaging experiments of co-cultures of MCF-7 hFWEKO cells expressing the four hFWE isoforms. In [1], MCF-7 hFWEKO cells expressing hFWE1–IRES–GFP were co-cultured with cells expressing hFWE1–IRES–RFP. GFP+ and RFP+ cells were monitored at 0 and 24 h to follow the effects of hFWE isoforms on cell proliferation. The ratio of GFP+ to RFP+ cells did not vary significantly between 0 and 24 h. In co-culture experiments [2], MCF-7 hFWEKO cells expressing hFWE1–IRES–GFP were co-cultured with cells expressing hFWE2–IRES–RFP. The population of RFP+ cells was significantly higher at 24 h than at 0 h, indicating competition between hFWE1–IRES–GFP and hFWE2–IRES–RFP cells. Each co-culture combination is presented amongst the four hFWE isoforms along with IRES–GFP or IRES–RFP co-expression. Cells expressing hFWE2 or hFWE4 emerged as winners when co-cultured with cells expressing hFWE1 or hFWE3 regardless of GFP or RFP reporter. The ratio of GFP+ to RFP+ cells at 0 and 24 h for each co-culture experiment is presented quantitatively below. The ratios at 0 and 24 h for each combination were compared statistically using a two-tailed t-test assuming unequal variances; n = 4 biologically independent experiments, P values shown, mean ± s.d. b, Analysis of live-cell imaging is shown as heat maps to represent the total number of cells, the number of cells that died every 1.5 h, average cellular volume, and average cellular sphericity. The co-culture combinations are indicated on the left. For example, the co-culture combination of cells expressing hFWE1–IRES–GFP with cells expressing hFWE2–IRES–RFP results in the death of hFWE1–IRES–GFP cells at the expense of an increase in the number of hFWE2–IRES–RFP cells. Analysis of all co-culture combinations is presented. The data support the idea that cells expressing hFWE1 or hFWE3, when co-cultured with cells expressing hFWE2 or hFWE4, undergo cell death accompanied by loss of differentiated cellular architecture, indicated by decreased cellular volume (blue) and increased cellular sphericity (red). Each block represents a gradient scale of low (blue), medium (yellow) and high (red) for the number, size or shape of the cells (n = 4, analysis performed using the manual tool from Fiji and automated tools from Imaris, Genie tool used for representing data as heat maps).
