A) Rosa26-driven MycER^T2 expression in R26^lsl-MER MEFs is dependent upon Cre recombinase-dependent and is transgene dose-dependent. R26^lsl-MER/WT MEFs were infected with retrovirus driving Cre recombinase (+) or control vector (−) and selected with puromycin for 3 days. Western blotting of whole cell lysates with anti-human ERαantibody reveals expression of the predicted 92Kda MycER^T2 fusion protein. The asterisk denotes a non-specific band.
B) Simultaneous comparison of levels of endogenous Myc and MycER^T2 in wt, R26^MER/wt and R26^MER/MER MEFs. MEFs were isolated from embryos of each genotype and cultured in complete growth medium/10% FBS. Equal numbers of cell equivalents from each lysate were western blotted with SC42 anti-Myc antibody, detecting an epitope common to endogenous Myc and MycER^T2. Extracts from serum-deprived wt MEFs and from wt MEFs 2 hrs after addition of fresh medium were probed alongside, together with an equivalent number of HeLa cells, which over express c-Myc (~30,000 molecules per cell) for comparison (Moore et al., 1987).
C) Dose dependent expression of MycER^T2 mRNA driven from the Rosa26 locus. Q-PCR quantitation (mean + SEM) of MycER^T2 mRNA isolated from in wt, R26^MER/WT and R26^MER/MER MEFs, all normalized to GUS (n=3).

A) Organs from adult R26^MER/MER mice treated for 3 days with tamoxifen (tam) (n=7) or oil carrier (n=6), assessed for proliferation (Ki67 staining), and apoptosis (TUNEL assay). Activation of MycER^T2 drives abundant ectopic cell cycle entry in multiple tissues (exocrine and endocrine pancreas, kidney, liver, lung and colon are presented). However, with the exception of the colon (inset), no tissue exhibited concomitant Myc-induced apoptosis. Scale bars = 25 μm.
B) Quantification (mean + SEM) of Ki67 staining in colon sections from tamoxifen-treated R26^WT/WT (n=4), R26^MER/WT (n=6), R26^MER/MER mice (n=3), and from R26^MER/MER mice treated with carrier (n=3). T Test analysis indicates that the increase in proliferation from R26^WT/WT to R26^MER/WT is significant (* P = 0.001) as is the increase from R26^MER/WT to R26^MER/MER (** P = 0.004).
C) Quantification (mean + SEM) of TUNEL positive cells in sections of colonic epithelium from same mice as B).

A) Q-PCR analysis of MycER mRNA expression in islets isolated from disaggregated pancreata of untreated designated mice, pooled according to genotype (n>2 per cohort). Expression was normalized to GUS and results are expressed relative to MycER^T2 mRNA in R26^MER/MER islets.
B) TUNEL staining of pancreatic islets from R26^MER/MER mice, treated with tamoxifen for 3 (n=7) or 6 (n=6) days (center panels), with matched samples from similarly treated pIns-MycER^TAM mice (lower panels). Ki67 staining of adjacent sections from R26^MER/MER pancreata (upper panels) demonstrates tamoxifen-induced functionality of Rosa26-driven MycER^T2 protein.

A) Representative images of H & E-stained lungs from tamoxifen treated, Ad-Cre infected R26^{lsl-MER/lsl-MER} mice showing multiple pre-neoplastic pulmonary lesions. Panels 1, 2: Localized epithelial dysplasia (ed) evident in airway epithelium (panel 1) and bronchioalveolar junctions (panel 2) after 3 weeks of sustained MycER^T2 activation. Compare with normal bronchiolar epithelium (n). Scale bars=20 μm. Panels 3, 4: Micro-papillary invaginations (panel 3) and atypical Clara cell proliferation characterized by vertical stacking of bronchiolar epithelial cells with apical nuclei (panel 4) present after 6 weeks of sustained MycER^T2 activation. Scale bars=40 μm.
B) H & E stained lungs from Ad-Cre infected LSL-K-Ras^G12D;R26^{lsl-MER/lsl-MER} mice treated with oil carrier (n=3, panels 1–4) or tamoxifen (n=6, panels 5–12) for 6 weeks. Panels 1, 2, 5 & 6: Size comparison between tumors driven by K-Ras^G12D alone (1, 2) and K-Ras^G12D/MycER^T2 combined (5, 6). Scale bars=0.4 □m (1, 5) and 100 μm (2, 6). The numbered rectangular regions within panels 1, 3, 5, 6, 7 and 9 are shown enlarged in panels 2, 4, 6, 11, 8, and 10 respectively. Panels 3, 4, 7 & 8: Micropapillary formation along the bronchioles driven by K-Ras^G12D alone (3, 4) and K-Ras^G12D/MycER^T2 combined (7, 8). The arrowhead points to a mitotic figure in panel 8. Scale bars=50μm (3, 7) and 20 μm (4, 8). Panels 9–11: Progression to carcinoma observed after 6 weeks of combined Kras^G12D/MycER^T2 driven oncogenesis. Scale bars=100 μm (9) and 20 μm (10, 11, 12). Panel 10: Nests of solid tumor (st) and isolated tumor cells surrounded by desmoplastic stroma (ds). Panels 11, 12: Mitotic figures (arrowheads) present in combined K-Ras^G12D/MycER^T2-driven papillary adenoma (pa), adenocarcinoma (ac; 11) and acinar adenocarcinoma.

A) Q-PCR analysis of MycER^T2 mRNA levels (mean + SEM) in selected organs from untreated adult R26^{lsl-MER/lsl-MER} (white bars), R26^MER/WT (grey bars) and R26^MER/MER (black bars) mice, normalized to GUS (n=3).
B) Representative images of 2-colour immunofluorescent detection of BrdU/IdU incorporation, indicating instances of S-phase progression in mice treated daily with tamoxifen for 6 days. Yellow-Orange: BrdU incorporation (S phase on day 3); Green: IdU incorporation (S phase on day 6).
C) Quantification (mean + SEM) of BrdU incorporation in tissues of control, R26^MER/wt and R26^MER/MER mice at day 6.

A) Q-PCR analysis for expression of ARF mRNA in organs from adult R26^MER/MER mice treated for 3 days with tamoxifen (n=7) or oil carrier (n=7), normalized to GUS. No ARF is induced by MycER^T2 activation in any tested tissue except for colonic epithelium, where it is statistically significant (* P value = 0.004).
B) Representative 2-colour immunoflourescence images of tissues from R26^MER/MER;ARF^+/+ (n=7) and R26^MER/MER;ARF^−/− (n=4) mice, injected daily with tamoxifen for 6 days. Mice were systemically pulsed with BrdU on day 2 and IdU on day 6, as per Figure 2, to identify cells in S phase at day 2 (Yellow-Orange) and day 6 (green).
C) Quantification (mean + SEM) of BrdU incorporation on day 6. Note that the data for ARF^+/+ are the same as those presented in Figures 2C.
D) Quantification (mean + SEM) of Ki67 staining of colon sections from the indicated mice treated for 3 days with tamoxifen. Data for ARF^+/+ samples are the same as those presented in Figure 3B. R26^WT/WT;ARF^−/− (n=3); R26^MER/MER;ARF^−/− (n=2).

A) TUNEL staining of pancreatic islets (panels 1, 2), liver (panels 3, 4), and colon (panels 5, 6) from R26^WT/WT (n=2) and R26^MER/MER (n=4) mice treated for 3 days with tamoxifen and overnight with 10 mg/kg doxorubicin.
B) Quantification (mean + SEM) of TUNEL staining in sections from pancreas (islets), liver and colon from the above mice treated with tamoxifen and/or doxorubicin. (n=2–4 mice per cohort).