Supplementary Figure 3: Further characterization of HGD lesions.

(a) Effect of DEN and Sorafenib treatment on differentiation. Confocal image of cyrosection from lesion stained for terminal differentiation marker FLG (green), KRT4 (magenta) and basal cell marker KRT14 (white). Scale bar, 200 μm. (b) Confocal image of cryosection stained for keratinocyte stress induced protein KRT17 (green) and basal cell marker ITGA6 (white). Scale bar, 200 μm. Images a,b are representative of 3 lesions. (c)Immune infiltrate in stromal wholemount stained for pan leukocyte marker CD45 (green) and KRT14 (magenta) marking lesion, Scale bar, 100 μm. Image representative of at least 3 tumours. (d–g) Clonal origin of HGD lesions. (d) Protocol. In Ahcre^ERTRosa26^flYFP/wt mice YFP was induced and animals then treated with DEN and Sorafenib to induce lesions. Possible outcomes are shown. Lesions derived from a single cell would be either unlabelled or completely labelled, while the presence of multiple labelled and/or unlabelled areas indicates multiple cells of origin. (e) Oblique view of 3D rendered confocal Z stack of typical lesion, dotted white line indicates lesion edge, YFP is green, KRT14 magenta. Scale bar, 200 μm. Projected confocal optical sections of typical lesion, YFP is green, KRT14 is magenta, ITGA6 is white. Scale bar, 100 μm. Images in e are representative of 14 lesions. (f) YFP + cell clusters per lesion (n = 18 lesions from 6 animals). Line indicates mean. (g) Area of YFP + clusters (n = 51 clusters from 6 lesions). Line indicates mean. See Supplementary Table 4 for source data for f and g.