JCS021147 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Upper panel: reverse transcriptase/PCR analysis of HK1.Fos expression identifies ∼700 bp v-fos-specific band in: non-phenotypic normal HK1.Fos adult skin (lane N; N*: w/o reverse transcriptase control for DNA contamination) and RU486-treated HK1.Fos/Δ5Pten ^flx or HK1.Fos/Δ5Pten ^het KAs. Lower panel: Δ5Pten expression in: untreated skin (lanes 1 and 2) and RU486-treated skin from HK1.Fos/Δ5Pten ^Het (lanes 3 and 4) or HK1.Fos/Δ5Pten ^flx (lanes 5 and 6) identify a Δ5Pten floxed allele-specific band (∼1100 bp), the wild-type allele (900 bp) and the truncated Δ5Pten floxed allele-specific band following exon 5 ablation in treated keratinocytes (400 bp).

• Supplemental Figure S2 -

  Fig. S2. Composite micrographs of protein expression in HK1.Fos/Δ5Pten^flx keratoacanthomas. Adjacent sections are those shown in Fig. 6 and in each composite the left panel/area shows the proliferative, papillomatous histotype, while the right displays differentiated frond regions. Both p53 and p21^WAF exhibit little expression in the papillomatous areas; however, strong increasingly basal layer expression appeared in differentiated/keratotic areas, with p21^WAF expression appearing in both cytoplasm and nucleus. P-GSK3β expression increased in papillomatous histotypes prior to the p53/p21 expression burst, but this expression remained essentially suprabasal, whereas in differentiated histotypes, a further increase and basal layer P-GSK3β expression was observed. Conversely, an inverse relationship was recorded for P-AKT expression. Strong expression in papillomatous regions faded in the corresponding p53/p21^WAF-expressing differentiated histotypes. A more-subtle finding in transitional areas demonstrated that strong P-AKT co-expression produced a less intense and suprabasal p53/p21^WAF expression profile and vice versa, increasing p53/p21^WAF expression produced less intense and suprabasal P-AKT co-expression, until P-AKT expression faded. Scale bar: 150 µm.

• Supplemental Figure S3 -

  Fig. S3. Immunohistochemical analysis of p53, p21^WAF, P-GSK3β and P-AKT expression in HK1.Fos and K14.cre/Δ5Pten histotypes. HK1.Fos and K14.cre/Δ5Pten sections are from control siblings age matched to those shown in Fig. 6 andFig 2S. In both HK1.Fos papillomas and K14.cre/Δ5Pten epidermal hyperplasia, p53 and p21^WAF expression were consistently negative, as were normal and hyperplastic HK1.Fos epidermis (not shown). In normal or early hyperplastic HK1.Fos epidermis, P-GSK3β levels were low (not shown), whereas in later hyperplasias/early papillomas, P-GSK3β expression increased to detectable levels, which remained suprabasal and similar but below to papillomatous HK1.Fos/Δ5Pten histotypes (Fig. 6I). Conversely, hyperplastic HK1.Fos epidermis possessed very little P-AKT expression, which remained low in papillomas (not shown). Hyperplastic K14.cre/Δ5Pten epidermis displayed strong P-AKT expression in each epidermal compartment, including basal layers and follicles, yet no papillomas appeared and hyperkeratosis was the predominant histotype. This was mirrored by P-GSK3β in being a downstream target of AKT, but P-GSK3β expression was mainly suprabasal and below that observed in HK1.Fos/Δ5Pten KAs (Fig. 6). Scale bars: 50 or 100 µm.