(A) Schematic showing Akt1-GFP-HRS cell line. HRS cells were modified to express a myristoylated fusion protein that includes the activated form of Akt1 fused to a mutant version of the ligand binding domain of the murine oestrogen receptor (mER). This mutant ligand binding domain recognizes 4-Hydroxy-tamoxifen (Tam) in place of oestrogen and places Akt1 under the control of Tam. (B) Western blot showing endogenous and conditional transgenic Akt1 expression in response to Tam and LY treatment. Differentiating cells were harvested on d4, 1 hr after Tam/LY treatment. (C) Flow cytometry showing uniform expression of the Akt1 containing transgene in Akt1-GFP-HRS cells during endoderm differentiation. Akt1-GFP-HRS cells were compared to HRS on day 6 of differentiation. (D) Western Blot confirming the Tam-induced expression of the Akt1 trangene in the GFP+ fraction of mixed HRS and Akt1-GFP-HRS co-cultures following cell sorting by flow cytometry based on GFP. Endogenous pAKT1 expression was observed in control conditions and was blocked by LY treatment. Cells were sorted based on GFP expression 6 hr after LY and Tam+LY (T/LY) treatments. (E) Quantification of the rescue of ADE induction as assessed by flow cytometry in Figure 5D. The experiments were performed by mixing HRS cells with Akt1-GFP-HRS cells (1:1), differentiating in the presence of LY and Tam, and assessing HRS expression in the different GFP populations at day 6. Blue columns represent the percentage of HRS expressing cells in the GFP− fraction (non-Akt1 expressing cells). Red columns represent the GFP+ fraction (Akt1 expressing cells). Treatments are stated below each set of columns. For normalization, HRS expression in control mixed cultures was taken as 100% for each fraction. Quantification of the non-cell autonomous effect of Tam was obtained by the ratio between HRS+GFP−/GFP−, while similar quantification of the cell-autonomous effect was obtained by the ratio between HRS+GFP+/GFP+, and compared to the control. LY decreased HRS expression in both GFP− (14.76% +/− 8.12) and GFP+ (21.68% +/− 7.26) fractions, whereas in Tam/LY treated cultures rescue of HRS expression was observed in both GFP− (65.95% +/− 21.87), and GFP+ (81.66% +/− 14.03) fractions, (n = 4).