Figure 3: Application of Fluoppi to quantitative analysis of drug-induced PPI blockage.

(a,b) High-content analysis (HCA) approach to the quantification of nutlin-3-induced dissociation of p53/MDM2 complex in CHO-K1 cells stably co-expressing PB1-p53 and AG-MDM2. After treatment of various concentrations of nutlin-3 for 30 min, cells were fixed with 4% PFA for 10 min and their nuclei were stained with Hoechst33342. (a) Domain structures of transfected constructs and two representative images of cells treated with 0.3 and 40 μM nutlin-3. (b) Dose (nutlin-3 concentration)-response (normalized P.I.) curve. Both puncta and nuclei were segmented automatically. P.I. was obtained by dividing the total AG (green) fluorescence from all the puncta by the total number of nuclei in each field of view. Normalized to the P.I. value at the lowest concentration.(c,d) High-throughput screening (HTS) approach to the quantification of nutlin-3-induced dissociation of p53/MDM2 complex in CHO-K1 cells stably co-expressing PB1-p53 and AG-MDM2. After treatment of various concentrations of nutlin-3 for 30 min, cells were fixed and permeabilized with PBS(−) containing 0.75% PFA and 2% Triton X-100 for 10 min and their nuclei were stained with Hoechst33342. (c) Domain structures of transfected constructs and two representative images of cells treated with 0.3 and 40 μM nutlin-3. (d) A dose (nutlin-3 concentration)-response (normalized P.I.) curve. No segmentation was performed. P.I. was obtained by dividing the total AG (green) fluorescence by the total Hoechst33342 (blue) fluorescence for each field. Normalized to the P.I. value at the lowest concentration. (e–g) Quantification of AT-406-induced dissociation of Smac/XIAP complex in HEK293 cells stably co-expressing SmacNT-PB1 and XIAP-AG. (e) Domain structures of transfected constructs are depicted (leftmost). Fluorescence images of cells 5 min before and 5 and 10 min after the addition of 25 μM AT-406 are shown (middle). Puncta were segmented automatically, and cell number was counted manually. The time course of averaged P.I. is shown (rightmost). See also Supplementary Video 6. (f) Domain structures of transfected constructs and two representative images of cells treated with 0.05 and 100 μM AT-406. (g) Dose (AT-406 concentration)-response (normalized P.I.) curve. Both puncta and nuclei (Hoechst33342-stained) were segmented automatically. P.I. was obtained by dividing the total AG (green) fluorescence from all the puncta by the total number of nuclei in each field of view. Normalized to the P.I. value at the lowest concentration. (a–g) Each experiment was performed in triplicate. Scale bars, 100 μm. Scale bars in magnified boxes, 10 μm. See also Supplementary Fig. 3.