Fig. 7: In vivo-optimized CeTEAM PARPi-GFP biosensors for ex vivo detection of drug-target engagement.
From: Coupling cellular drug-target engagement to downstream pharmacology with CeTEAM
a The PARP1 L713F-GFP biosensor was paired with mCherry for normalization. b Representative fluorescent micrographs (from n = 3) of U-2 OS PARP1 L713F-GFP/mCherry cells treated with DMSO or 1 µM veliparib for 24 hours. Scale bar = 50 µm. c mCherry-normalized L713F-GFP signal from 24-hour veliparib by flow cytometry. Modal normalization is shown. Representative of n = 2. Gray-blue gradient – veliparib gradient. d Graphical overview of in vivo experiments with HCT116 subcutaneous xenografts constitutively expressing PARP1 L713F-GFP and mCherry treated with either niraparib or vehicle control (2x, qd). nVehicle: 4, n15mg/kg: 3, n60mg/kg: 4 mice per treatment group. e Representative flow cytometry histograms of PARP1 L713F-GFP/mCherry tumors. Modal normalization is shown. Veh4: vehicle (mouse #4; gray), 15mg3: 15 mg/kg (mouse #3; blue), and 60mg4: 60 mg/kg (mouse #4; orange). f mCherry-normalized L713F-GFP intensity of tumors by flow cytometry. Means with 95% confidence intervals are shown from n = 3 (15 mg/kg) or n = 4 mice (Vehicle, 60 mg/kg). g Gross L713F-GFP signal from individual tumors by western blot. h mCherry-normalized L713F-GFP abundance from western blots in g and Supplementary Fig. 19e. Means with 95% confidence intervals are shown from n = 3 (15 mg/kg) or n = 4 mice (Vehicle, 60 mg/kg). i Representative L713F-GFP micrographs from tumor sections with fire LUT pixel density depiction. Scale bars=100 µm. j Floating histogram of L713F-GFP intensities across tumors and treatment groups (gray – vehicle, blue – 15 mg/kg, orange – 60 mg/kg). Blue region represents an arbitrary cut-off of GFP intensity ≥ 0.075 RFU. 16,482 total cells per treatment group. k Distribution of individual cell L713F-GFP intensities ≥0.075 RFU. Violin plots with median (thick line) and quartiles (thin lines) are overlayed onto individual datapoints. In all cases, P values are shown for one-way ANOVA (Dunnett’s test; f and h; FTreatment (f) [DFn, DFd] = 32.64 [2, 8], FTreatment (h) [DFn, DFd] = 17.384 [2, 8]) or Kruskal-Wallis test (Dunn’s test; k; Kruskal-Wallis statistic = 160.0) with multiple comparisons to the vehicle control. RFU relative fluorescence units.
