Inhibition of the PI3K-Akt pathway impairs Notch signalling in Jurkat T-cells. (A) J-N2 cells were transfected with 14 μg of p10xCBF1-luc and 1 μg pTK-RL, incubated overnight, then plated onto 96-well plates coated with either 20 μg/ml hIgG4 or 20 μg/ml hDelta1-Fc along with LY294002 for 6 hours. CBF1-reporter gene activity is normalised to Renilla control in this and all luciferase assays. (B) J-N2 cells were plated onto either hIgG4 or Delta1-Fc ± 10 μM LY294002, and incubated for 8 hours before being processed for Hes1 mRNA levels by Q-PCR (Hes1/18 rRNA). (C) J-N2 cells were transfected with 10 μg of pCBF1x10-luc, 4 μg of Pten expression construct or empty pcDNA3.1 vector,1 μg of pTK-RL, and treated with either 20 μg/ml hIgG4 or 20 μg/ml hDelta1-Fc ± 10 μM LY294002. CBF1-reporter activity was measured after 6 hr. All data is presented as mean ± s.d., and is representative of three repeat experiments. (D) J-N2 cells were transfected with p10xCBF1-luc and pTK-RL and stimulated as described in Fig. 1A, except that cells were treated with Akt inhibitor SH-6 (50 μM) or DMSO vehicle for 6 hours.