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

Figure 4.

Figure 4

Concomitant induction of apoptosis and FoxO3a expression by dose-dependent suppression of canonical Notch signalling. (A) Primary human keratinocytes were transfected with the Notch-pGA reporter (pGA-Luc; 0.5 μg) together with the phRL-TK Renilla reporter and subsequently infected with an adenovirus expressing the MAM51 peptide or GFP control at the indicated MOI. Promoter activity was measured 24 h later. Results are expressed as a ratio of luciferase activity (after Renilla normalization) in cells infected with the AdMAM51 versus AdGFP viruses at the various MOIs. (B) Human primary keratinocytes, together with keratinocyte-derived cancer cell lines (SCCO12, SCCO22, HeLa and CasKi), were infected with AdGFP and AdMAM51 at the indicated MOI. The fraction of apoptotic cells was assessed 24 h later by TUNEL assays. (C) Human primary keratinocytes and the SCCO12 and SCCO22 cell lines were infected with the AdMAM51 and AdGFP viruses at the indicated MOI and analysed 24 h later for levels of FoxO3a mRNA by real-time RT–PCR. Values are expressed in arbitrary units after normalization for β-actin expression. (D) Human primary keratinocytes were infected with AdMAM51 and AdGFP at an MOI of 100, followed by nuclear and cytoplasmic fractionation and immunoblot analysis for the FoxO3a protein, using the β-actin and TBP proteins as equal loading controls for the cytoplasmic and nuclear fractions, respectively. (E) Human primary keratinocytes were infected with AdMAM51 and AdGFP at an MOI of 100 as before. Cells were fixed 24 h later and processed for immunofluorescence analysis with an antibody against FoxO3a. (F) Human primary keratinocytes were transfected with siRNA specific for CSL in parallel with scrambled siRNA control, followed by assessment of CSL expression at 24, 48 and 72 h after transfection by real-time RT–PCR. (G) Parallel cultures treated as in panel F were analysed by TUNEL assays at various times (hours) after CSL knock-down. (H) Keratinocytes with and without CSL knock-down as in the previous experiments were analysed for levels of FoxO3a expression by real-time RT–PCR and immunoblotting (inset). (I) Primary human keratinocytes were infected with adenoviruses expressing activated Notch1 or GFP control for 24 h and analysed for levels of FoxO3a mRNA by real-time RT–PCR. (J) Primary human keratinocytes were transfected with a FoxO3a-responsive reporter (FHRE-luc) plus increasing amounts of an expression vector for activated Notch1. Promoter activity was determined at 30 h after transfection by luciferase assays, using the phRL-TK Renilla reporter for internal normalization. (K) Primary human keratinocytes infected with adenoviruses expressing activated Notch1 or MAM51 in parallel with GFP controls were analysed by real-time RT–PCR for levels of expression of Bcl6 and Bim1, two well-established FoxO3a targets with pro-apoptotic function.