Article Title: Defining the clonal dynamics leading to mouse skin tumour initiation
Figure Lengend Snippet: Clonal dynamics of interscale K14-CreER/Rosa-SmoM2 clones is consistent with two populations (a) Distribution of K14-CreER/Rosa-YFP (black) and K14-CreER/Rosa-SmoM2 (red) clone sizes as measured by total cell content, imaged by confocal microscopy on whole mount tail epidermis from 1w to 24w post-induction. The number of clones analyzed for K14-CreER/Rosa-SmoM2 is indicated in Fig 4b; the number of clones counted in K14-CreER/Rosa-YFP is as indicated in Fig. 2a. (b) Evolution of the clonal persistence and (c) labelled cell fraction for K14-CreER/Rosa-SmoM2 clones in the interscale. (d) Fraction of EdU/BrdU double-labelled cells as a function of basal clone size at 8w for K14-CreER/Rosa-SmoM2 clones, for 2 (left), 4 (centre) and 6 (right) days of continuous BrdU incorporation. (e) Immunostaining for β4-integrin, SmoM2 and active-caspase-3 in K14-CreER/Rosa-SmoM2 clones 8w after SmoM2 activation. (f) Percentage of BCC, dysplastic, hyperplastic and normally differentiating clones presenting at least one active-caspase-3 positive cell at 8w post-induction (n=117 clones analysed from 4 independent experiments). (g) Quantification of the number (%) of basal and suprabasal apoptotic cells in dysplastic, hyperplastic and normally differentiating Inv-CreER/Rosa-SmoM2 clones 8w after SmoM2-activation. (h) Percentage of dysplastic, hyperplastic and normally differentiating Inv-CreER/Rosa-SmoM2 clones presenting basal and suprabasal apoptosis 8w after oncogenic activation. (i) Cumulative distribution of the fraction of basal apoptosis as a function of basal cell number in a K14-CreER/Rosa-SmoM2 clone at 8w (data in red). The green line is the expected theoretical distribution of apoptotic fraction if apoptosis occurred randomly (following a Poisson process), in any clone with the same probability. The data is statistically different from the random theory, showing that apoptosis clusters in certain clones at a given time point. (j) Quantification of EdU/BrdU double-labelled cells as a function of the period of continuous BrdU incorporation for large K14 clones at 4w (black), 8w (orange) and 12w (red) post clonal induction. The dashed lines represent the model fit ( Supplementary Theory ). (k) Whisker plot of the suprabasal clone size in the interscale. The boxes delineate the first and third quartiles of the data, while the whiskers delineate the first and last decile of the data at a given time point. The thick continuous line is the best fit from the model from which we extract the probability of fate choices in tumor SC and progenitors, displayed in Fig.4g . The thin lines represent the mean clone sizes of stem cells (top curve) and progenitors (bottom curve) if they were alone. (l) Short-term fate outcome of progenitors in K14-CreER/Rosa-SmoM2 clones at 8w, as assessed by using EdU as a clonal marker. We count only cell doublets and classify them as either basal-basal, basal-suprabasal, or suprabasal-suprabasal. (n=49 clones from 3 independent experiments) Immunostaining for β4-integrin, EdU and SmoM2 in K14-CreER/Rosa-SmoM2 hyperplastic/dysplastic clones (top) and in BCC (bottom panel). BCC: basal cell carcinoma; D: dysplasia; H: hyperplasia; N: normal differentiation; SB: suprabasal. Hoechst nuclear staining is represented in blue; Scale bars, 10μm. Error bars represent the s.e.m.
Article Snippet: Primary antibodies used were the following: anti-GFP (Rabbit, 1/100, BD, ref. A11122), anti-GFP (Goat, 1:800,Abcam, ref. Ab6673), anti-active-caspase3 (Rabbit, 1/600,R & D, ref. AF835), anti-β4-integrin(Rat, 1:200, BD, ref. 553745) and anti-K31 (Guinea Pig, 1:200, Progen, ref. GP-hHa1).
Techniques: Clone Assay, Confocal Microscopy, BrdU Incorporation Assay, Immunostaining, Activation Assay, Whisker Assay, Marker, Staining