Figure 3: Tamoxifen-regulated FlpO-ER efficiently restores p53^TR alleles to p53^Rin vitro and in vivo.

(a) GFP detection by flow cytometry analysis of KP^XTR/XTR; Rosa26^FlpO-ER/+ MEFs. Untreated (p53^XTR/XTR: solid grey), AdCre treated (p53^TR/TR: solid green), tamoxifen-treated cells previously untreated (p53^R*/R*: open black trace) or previously AdCre-treated (p53^R/R: open green trace). R*, direct conversion of p53^XTR to p53^R. (b) Analysis of transformation potential of KP^XTR/XTR; Rosa26^FlpO-ER/+ MEFs after in vitro AdCre treatment followed by subcutaneous engraftment into nude mice. Tumour growth monitored by caliper measurements at the indicated times. Tamoxifen was administered on day 0, 1 and 2 to all mice. Representative of two cell lines (n=4). (c) Immunoblot analysis of lysates from KP^TR/TR; Rosa26^FlpO-ER/+ lung tumour-derived cell lines 24 h after addition of 4-hydroxytamoxifen (4-OHT) or vehicle control. (d) Immunoblot analysis of lysates from micro-dissected lung tumours 7 days after tamoxifen delivery. Immune precipitation of p53 was required before western blotting for detection. (e) Microscopic analysis of lung tumours derived from KP^XTR/XTR; Rosa26^FlpO-ER/+ and KP^XTR/XTR; Rosa26^+/+ mice 7 days post tamoxifen treatment. From left to right, bright-field and fluorescence stereo microscopy, haematoxylin and eosin staining and immunohistological staining for GFP in representative tumours. Scale bars, 100 μm.