Figure 4: Monitoring clonal evolution during metastasis.

(a–h) Mice were injected i.v. with the MDA-MB-231 cell mixtures GLuc⁺nsh/CLuc⁺nsh, GLuc⁺shp53.1/CLuc⁺nsh or GLuc⁺shp53.5/CLuc⁺nsh. (a) Bioluminescence in vivo imaging of GLuc activity with coelenterazine. (b) GLuc (coelenterazine) and CLuc (vargulin) imaging of explanted lungs. (c) GLuc/CLuc activity ratios in lung lysates (mean±95% confidence interval; *P<0.01, nonparametric Kruskal–Wallis test and Dunn’s post test for multiple comparisons). (d) Western blot for knockdown efficiency of experimental shRNAs. (e) Immunohistochemical double staining for p53 (brown) and GLuc (red) in lungs explanted from mice injected with (left) GLuc⁺nsh/CLuc⁺nsh, (middle) GLuc⁺shp53.1/CLuc⁺nsh, and (right) GLuc⁺shp53.5/CLuc⁺nsh cell mixtures. Scale bars, 100 μm. (f–h) GLuc/CLuc plasma activity (mean±s.e.m.). Tumour growth curves were analysed by two-way analysis of variance (*P<0.005).