Fig. 3: Sequence of intermediates during reprogramming and transformation.
a, Representation of Bcl11b and Thy1 expression in single cells. The thresholds were as follows: Bcl11b < 1 and Thy1 < 2 for BLTL; Bcl11b > 2 and Thy1 < 2 for BHTL; Bcl11b < 1 and Thy1 > 4 for BLTH; and Bcl11b > 2 and Thy1 > 4 for BHTH. b, Expression of Bcl11b-tdTomato and Thy1 during reprogramming and transformation. KI: knock-in. c, Left, pictures of iPS colonies from a representative experiment. Right, quantification of AP+ colonies (n = 6 independent experiments). d, Left, pictures of foci assays from a representative experiment. Right, foci quantification (n = 5 independent experiments). e, Emergence of Bcl11b-tdTomatolow/Thy1low cells. The graph represents the distribution of BHTH, BLTH, BHTL and BLTL cells. IWP2, Wnt inhibitor; OSKM, Oct4, Sox2, Klf4, c-Myc; SNEL, Sall4, Nanog, Esrrb, Lin28. BHTH cells were FACS sorted before reprogramming/transformation. f, Emergence of BLTL cells from mouse adult ear fibroblasts. The settings were similar to those for e. g, Left, pictures of iPS colonies following reprogramming induced by OSK + IWP2, taken from a representative experiment. Right, quantification of AP+ colonies (n = 2 independent experiments). h, Left, pictures of iPS colonies from mouse adult ear fibroblasts, taken from of a representative experiment. Right, quantification of AP+ colonies (n = 3 independent experiments). i, Schematic of the experimental design. j, Left, brightfield images of tumours in a chick (as indicated by the dashed lines). Right, quantification of the tumours (n = 23 for T-BLTL cells; n = 27 for T-BHTH cells). k, Left, tumour growth curves. Right, survival curves of the mice (n = 6 animals per group). l, Left, FACS profiles. Cells harboring various levels of Bcl11b and Thy1 were FACS sorted at day 5 of reprogramming, plated back in culture and analyzed 2 days later. Right, quantification of AP+ colonies (n = 5 independent experiments). m, Left, FACS profiles. Cells harboring various levels of Bcl11b and Thy1 were FACS sorted at day 5 of transformation, plated back in culture and analyzed 2 days later. Right, quantification of foci (n = 6 independent experiments). n, Schematic of the sequence of intermediates. The corresponding efficiencies are indicated using arbitrary units. In h, j and the left panel of k, the data represent means ± s.d. Statistical significance was determined by two-tailed Student’s t-test (c, d, h and j), two-way ANOVA combined with Šidák’s multiple comparisons test (left panel in k), Gehan–Breslow–Wilcoxon test (right panel in k) or one-way ANOVA followed by Tukey’s post-hoc test (l and m).
