Figure 6.

The Notch protective function in the keratinocyte UVB response through down-modulation of FoxO3a expression. (A) Primary human keratinocytes infected with AdMAM51 and GFP control at 100 MOI for 24 h were either untreated or treated with the PI3K inhibitor wortmannin (100 nM) for an additional 8 h, followed by analysis of FoxO3a protein levels by immunoblotting with a corresponding specific antibody. (B) Primary human keratinocytes were stably infected with a MAM51-expressing retrovirus in parallel with a GFP control virus and either untreated or treated with UVB (50 mJ/cm²). Cells were analysed 24 h later for levels of total FoxO3a protein as well as protein phosphorylated at the critical Akt recognition site (Thr32) (pFoxO3a) by immunoblotting with the corresponding specific antibodies. The same extracts were also probed with antibodies against the phosphorylated active form of Akt (pAkt), in parallel with γ-tubulin as an equal loading control. (C) Similarly, retrovirally infected cells plus/minus UVB treatment as in panel B were analysed for FoxO3a mRNA levels by real-time RT–PCR. (D) Primary keratinocytes from Notch1^loxP/loxP mice were infected with AdCre, to induce deletion of the Notch1 gene, or AdGFP control, as in Figure 1. Cells were subsequently treated with UVB (50 mJ/cm²) followed, 30 h later, by measurement of FoxO3a mRNA levels by real-time RT–PCR. (E) Human primary keratinocytes were incubated for 16 h with DAPT (10 μM) or DMSO control, UVB-irradiated (50 mJ/cm²) and, 8 h later, either collected or treated with actinomycin D (8 μg/ml) for the indicated times (hours). FoxO3a mRNA levels were assessed by real-time RT–PCR. (F) Human primary keratinocytes were transfected with validated siRNAs for FoxO3a or scrambled controls, followed by measurement of FoxO3a mRNA levels 48 h later by real-time RT–PCR. (G) Human keratinocytes stably infected with the MAM51- and GFP-expressing retroviruses were transfected with FoxO3a siRNAs or scrambled siRNA controls followed, 48 h later, by UVB irradiation (50 mJ/cm²). The apoptotic response was determined 24 h after irradiation by TUNEL assays. (H) Similarly treated cells were analysed for levels of activated caspase 3, by immunoblotting with the corresponding antibodies, with γ-tubulin as an internal control. (I) Human primary keratinocytes were transfected with siRNAs for FoxO3a or scrambled controls and 24 h later treated with DAPT (10 μM) or DMSO control. Cells were UVB irradiated 24 h later and, after another 24 h, were analysed by TUNEL assays.