Figure 3.

In Vitro Visualization of Transfer of Cre Activity

(A) Images of MDA-MB-231 cultures consisting of reporter⁺ cells (red) only or a mixture of Cre⁺ (cyan) and reporter⁺ cells. Note the appearance of eGFP⁺ reporter⁺ cells in the presence of Cre⁺ cells, indicated by asterisks. Scale bars represent 50 μm.

(B) Quantification of the percentage of eGFP⁺ MDA-MB-231 cells in several conditions with varying ratios of Cre⁺ and reporter⁺ cells (n = 5 independent experiments). Data are represented as mean ± SEM.

(C) For 50 cells, the mean fluorescence intensity (MFI) of eGFP (reporting Cre activity) is plotted against the MFI of CFP (Cre⁺ cells). Intensities of non-fluorescent cells are zero (red dot). In the left plot, cell fusion was experimentally simulated by transfecting eGFP⁺ cells with CFP DNA. In the middle plot, communication was experimentally simulated by transfecting reporter⁺ cells with Cre DNA. The right plot shows the results of the experimental co-culture. The blue dotted line outlines cells that show cell fusion, and the green dotted line shows eGFP expression derived from cell-cell communication other than fusion.

(D) Images of MDA-MB-231 reporter⁺ cells (left), T47D reporter⁺ cells (middle), or MCF-7 reporter⁺ cells (right) at the bottom well of a transwell system in the absence (upper images) or presence (lower images) of MDA-MB-231 Cre⁺ cells in the upper well (n = 3 independent experiments). Bar graphs below show quantifications. Data are represented as mean ± SEM. Scale bars represent 50 μm.

(E) Quantification of T47D cells (upper graph) and MCF-7 cells (lower graph) that express eGFP in several conditions with varying ratios of Cre⁺ and reporter⁺ cells (n = 5 independent experiments). Data are represented as mean ± SEM.