Cell cycle following activation of MYC-ERTAM. A) Sections from 4OHT-treated MYC-ERTAM transgenic mice were stained with Ki67-specific antibodies (green) and a tissue specific antibody (red; insulin for β-cells, keratin 1 for SBK). DAPI staining (blue) identifies cell nuclei. No Ki67 staining was detected in β-cells or SBK in vehicle-treated samples, although proliferating basal cells in the skin showed clear staining. Following 32 hours of 4OHT, staining of both β-cells and SBK was detected, indicated by concomitant staining of Ki67 and the tissue-specific protein. Images shown are representative sections from different levels through replicate tissue samples. b, basal keratinocyte; sb, suprabasal keratinocyte. B) The change in expression of genes relating to cell cycle following activation of MYC-ERTAM is represented as a heatmap (red, up-regulated; black, no change; green, down-regulated). Significant change in expression is detected for key early G1-S phase transition genes such as Ccnd1, Ccnd2 and Ccne2, indicating activation of cell cycle progression, particularly for the pancreas. C) qRT-PCR validation for gene expression changes in genes relating to cell cycle progression indicated a clear proliferative response in the pancreatic β-cells following MYC-ERTAM activation. In particular, genes relating to G1/S-phase transition such as Ccnd1, Ccnd2 and Ccne2 showed early changes in expression (8 hours), whilst genes whose products relate to later cell cycle events such as Ccna2 and Ccnb1 showed increased expression at later time points (32 hours). Few significant changes were detected for these key proliferation markers in SBK. This may be due to the relatively low proportion of keratinocytes that are responsive to Myc-induced proliferation at these early time-points, whilst differentiated keratinocytes of the granular layer are refractory to the proliferative influence of MYC.