Skip to main content
. 2008 Apr 3;27(8):1243–1254. doi: 10.1038/emboj.2008.45

Figure 1.

Figure 1

Increased Notch1 signalling and expression in the keratinocyte UVB response. (A) Primary mouse keratinocytes were transfected with a synthetic Notch-responsive promoter consisting of six CSL binding sites in front of the luciferase gene (CSL-luc), followed, 24 h later, by UVB irradiation (50 or 100 mJ/cm2). Promoter activity was measured 24 h after irradiation by luciferase assays, using a Renilla reporter with a minimal promoter for internal value normalization. (B) Primary mouse keratinocytes were irradiated with UVB (50 mJ/cm2), followed, 2 h later, by measurement of HES1 mRNA levels by real-time RT–PCR. Values are expressed as relative units after internal normalization for GAPDH mRNA levels. (C) Back skin of 3 days old mice was irradiated with UVB (220 mJ/cm2). Eight hours later, the epidermis was separated from the underlying dermis by a brief heat treatment (Nguyen et al, 2006) followed by total RNA preparation and analysis of HES1 mRNA expression by real-time RT–PCR, using GAPDH mRNA for normalization. (D) Primary human keratinocytes transfected with siRNAs for p53 or scrambled siRNA controls for 48 h were irradiated with UVB (50 mJ/cm2) followed, 2 h later, by measurement of HES1, HERP1 and Notch1 expression levels by real-time RT–PCR, with 36B4 mRNA for normalization. Suppression of endogenous p53 was verified by real-time RT–PCR. (E) Primary human keratinocytes were treated as before and Notch1 protein levels were analysed at various times (hours) after irradiation by immunoblotting with the corresponding antibody, using blotting for γ-tubulin as an equal loading control. (F) RNA samples from the same cells as in panel D were analysed for levels of keratin 1 and involucrin differentiation marker expression by real-time RT–PCR with the corresponding specific primers. (G) Mid-back skin of healthy male volunteers, with their informed consent, was irradiated with an FL20S-E Lamp (290–320 nm) (Toshiba, Tokyo). Biopsies taken at the indicated times (days) were processed for immunostaining with antibodies specific for cleaved from of Notch1 (Val1744). Bar, 30 μm.