a, Quantification of the frequency (freq.) of Hes1pos cells in TKO tumours at different stages of tumour development. Scores: 0 (0%), 1 (1–20%), 2 (20–60%), 3 (>60%). Hyperplasias (n = 26, from five mice, defined by area less than 16,600 μm2) and early tumours (n = 69, from five mice) were analysed 3 months after Ad-CMV-Cre; late tumours (n = 83, from five mice) and liver metastases (n = 54, from five mice) were analysed 6–7 months after Ad-CMV-Cre (data for the late tumours are shown for comparison and are the same as in Fig. 1b). b, As in a, for human SCLC tumour microarray sections and segregated by clinical stage. H scores: stage I (123.9; n = 71 sections), stage II (135.3; n = 68 sections), stages III + IV (148.5; n = 33 sections). c, Representative immunofluorescence for GFP in TKO Hes1GFP/+ SCLC tumours. d, Representative flow cytometry plots of cells isolated from pooled tumours from a TKO Hes1GFP/+ mouse. Arrows depict the sequential gating strategy for enriching for single, live (by exclusion of 7-aminoactinomycin D (7-AAD)), and lineage (CD45, CD31, TER-119)-negative cells. CD24 labels more than 98% of Cre-recombined cells and thus further enriches for tumour cells39. e, Immunofluorescence of a TKO Rosa26lox-stop-lox-tdTomato;Hes1GFP/+ tumour section showing co-localization of GFP and Tomato signals. f, Flow cytometry shows that GFPhigh cells in pooled tumours from a TKO Rosa26lox-stop-lox-tdTomato;Hes1GFP/+ mouse infected with Ad-CMV-Cre are positive for Tomato expression (representative of n = 2 mice). g, Genotyping PCR analysis for recombination (delta, Δ) or the unrecombined (floxed) alleles at the Rb, p53, and p130 loci in GFPneg and GFPhigh tumour cells sorted from three TKO Hes1GFP/+ mice. DNA from fl/fl and/or Δ/Δ cells served as controls. Scale bars, 50 μm.