Figure 2

Protective function of Notch signalling in the UVB response of mouse keratinocytes and skin. (A) Primary mouse keratinocytes under growing conditions and at 24 h of calcium-induced differentiation (Missero et al, 1996) were treated with UVB (50 mJ/cm²) followed, 8 and 24 h later, by determination of the apoptotic response by TUNEL assays. (B) Primary keratinocytes from mice homozygous for the Notch1 gene flanked by loxP sites (Notch1^loxP/loxP) were infected with an AdCre, to induce deletion of the Notch1 gene, or AdGFP control. At 72 h after infection, part of the cells was induced to differentiate by calcium treatment. After 24 h, keratinocytes under either growing or differentiating conditions were treated with UVB (50 mJ/cm²). The apoptotic response was determined 12 h later by TUNEL assays. (C) Primary mouse keratinocytes plus/minus deletion of the Notch1 gene as in the previous panel were analysed, under growing conditions, by immunoblotting with antibodies specific for the caspase 3 cleaved form of PARP in parallel with antibodies against Notch1. (D) Mice with Cre-induced keratinocyte-specific deletion of the Notch1 gene (Notch1^loxP/loxP K5-Cre-PR1) in parallel with their Cre-negative littermates (Notch1^loxP/loxP) (Rangarajan et al, 2001) were irradiated with UVB (140 mJ/cm²) on their back skin, at 8 weeks of age. Apoptosis was measured 12 h later by TUNEL assays. (E) Primary mouse keratinocytes were infected with a recombinant adenovirus expressing a constitutive active form of Notch1 (AdNIC) or AdGFP control. After16 h, cells were UVB irradiated (50 mJ/cm²) and the apoptotic response was evaluated 8 h later by TUNEL assays. (F) Primary mouse keratinocytes were infected with AdNIC and AdGFP in parallel with recombinant adenoviruses expressing the CDK inhibitors p16^INK4a, p21^WAF1/Cip1 and p27^Kip1. After 16 h, cells were UVB irradiated (50 mJ/cm²) and the apoptotic response was evaluated 8 h later by TUNEL assays. (G) Primary mouse keratinocytes were infected with AdGFP and with the AdNIC virus individually and in combination with an adenovirus expressing a stabilized super-repressor mutant form of IB (IB-SR) (Wang et al, 1999). Cells were subsequently irradiated and analysed by TUNEL assays as in the previous panels.