Figure 2

Workflow of Fluoppi bimodal p53:Mdm2 and p53:Mdm4 PPI system. Separate CHO-K1 lines stably expressing either Ash-p53:AG-Mdm2 or Ash-p53:MR-Mdm4 Fluoppi PPI pairs were established and maintained independently. To enable bimodal measurement both cell lines were pre-mixed in equal proportions and then used to seed 96 well plates for compound analysis. Stably transfected cell lines were used rather than transiently transfected cells in order to reduce experimental steps and to allow finer control over the ratio of Ash-p53:AG-Mdm2 to Ash-p53:AG-Mdm4 derived foci. Plates were incubated overnight and cells were treated with PPI modulating drugs. High content analysis equipment (HCA) was used to obtain images of green (Mdm2), red (Mdm4) and blue (Hoechest 33342) channels, which were then processed and segmented into nuclei and fluorescent foci using the Harmony High Content Imaging and Analysis Software (PerkinElmer). Cells were also processed using a nuclear morphology filter. This information was used to calculate the PPI (FLUOPPI) signal in accordance with the formula displayed. A minimum number of 500 foci were used to determine this value. Dose curves for each compound were then derived and used to calculate a corresponding IC₅₀ value. A similar experimental setup can be used to analyse each stably transfected cell line separately.