Figure 4. B16xMEF hybrids stably maintain B16-restricted DNA, do not maintain expression of B16-Restricted GFP, and are hyperploid.

(A) Analysis of Cre DNA in B16-GFP-Cre, MEF, and twenty tdTomato⁺ clones by PCR. (B) Quantification of GFP expression in B16-GFP-Cre and twenty tdTomato⁺ clonal cell lines measured by FACS (n = 3 independent experiments). Data is represented as mean ± SEM. (C) FACS plot and fluorescent micrograph showing GFP and tdTomato expression in one of the three tdTomato⁺ clones (#D2) that showed loss of tdTomato expression. (D) Quantification of DNA content of B16-GFP-Cre and twenty tdTomato⁺ cell lines by FACS. Ploidy was determined by normalizing the 7-AAD MFI of each cell line relative to MEF, which was set at 2n (n = 3 independent experiments). Data are represented as mean ± SEM. See also Supplementry Figure 3. (E) Comparison of the average DNA content in ten B16-GFP-Cre clones and twenty tdTomato⁺ clones as determined by FACS (n = 2–3 independent experiments per data point). Data is represented as mean ± SEM. (F) Quantification of chromosome number in B16-GFP-Cre cells and eight tdTomato⁺ clonal cell lines by karyotyping (n = 15–20 metaphase spreads per group). Shown is mean ± SD. Representative metaphase spreads of B16-GFP-Cre and two tdTomato⁺ clones are shown on the right.