Figure 6

Titrations of the dual Mdm2/4 specific inhibitor VIP-82 and its scrambled negative control VIP-82^SCRAM against (A) CHO-K1 cells stably transfected with the bimodal FLUOPPI p53:Mdm2/4 system and (B) two independent cell populations either transiently transfected with the p53:Mdm2 nanoBIT system or the p53:Mdm4 nanoBIT system. Compound titrations were performed with either treatment periods of 4 hours or 24 hours. (C) CHO-K1 cells were treated with identical concentrations of VIP-82 and VIP-82^SCRAM as in (A,B) and their effects on viability were assessed at the same time points. (D) Bar charts indicate the relative number of cells used in the calculation of the FLUOPPI signal at each titration point indicated in (A). (E) Normalization of nanoBIT titrations results shown in B) to cell viability data in (C). Data points were removed at concentrations greater than 10 µM in the nanoBIT titrations normalized at 24 hours, due to substantial increases in luminescence values above the 1% DMSO v/v control. IC₅₀ values are indicated next to the relative titration. IC₅₀ values were derived from each individual titration using a 4-parameter curve model. Non-linear regression analysis to fit the curve was performed in Prism (Graphpad). Experiments were performed in DMEM cell media containing 2% (v/v) FCS