Figure 1

Schematic representation and construction of Fluoppi assay. (A) Mechanism of Action of Fluoppi. Ash tag and tetrameric fluorescence protein are fused to bait and prey proteins, respectively. Interaction between bait and prey (left panel, PPI+) proteins produces fluorescent foci inside mammalian cells via multivalent bait-prey interactions between single molecules. When the bait-prey protein interaction is inhibited by a PPI modulator (PPIM), FP tagged prey protein diffuse throughout the cells (Right panel, PPI−). Representative images of arbitrary Fluoppi expressing HEK293 cells are shown. Scale bar, 100 µm. (B) Representative images of selected Ash-p53:AG-Mdm2 and Ash-p53:MR-Mdm2 control pairs. Bright fluorescent foci were formed in the cytosol. (C) Representative images of selected Ash:AG-Mdm2 and Ash3:MR-Mdm4 negative control pairs. No Foci were detected with the negative Fluoppi control pairs, where either FP tagged Mdm2 or Mdm4 are co-expressed with the non-fused Ash tag by itself. (D) The reversibility of each of the systems were demonstrated either by treatment with an Mdm2 specific inhibitor (Nutlin-3A at 12.5 µM) or a dual inhibitor of both Mdm2 and Mdm4 (RO-5963 at 50 µM) at the following time points: 0 hour (pre-treatment), 1 hour or 24 hours. With Nutlin-3A, fluorescent foci mediated by the Mdm2:p53 interaction (green) but not the p53:Mdm4 (red) interaction were disrupted at both 1 hour and 24 hour treatment time points. RO-5963 only after 24 hours of treatment disrupted both sets of fluorescence foci meditated by both p53:Mdm2 or p53:Mdm4. Scale bar, 20 µm (B,C), 100 µm (D).