Fig. 5: Profiling of PARPi engagement and trapping in live cells with PARP1 L713F-GFP.

a Chemical structures and PARP1 inhibitory potencies of PARPi studied (SelleckChem and⁹⁴). b A representative blot (n = 2) of induced GFP-PARP1 WT and L713F-GFP in U-2 OS cells treated with PARPi for 24 hours. Black arrow – GFP-tagged PARP1; gray arrow – endogenous PARP1. c Experimental schematic for live cell tracking of PARP1 target engagement (GFP) and PARPi-induced replication stress (cell cycle, Hoechst) by high-content microscopy. Trapping depends on PARP1 engagement and replication stress. d Curve fitting of median GFP (solid) and Hoechst (DNA content; open/dashed) intensities in live, PARP1 L713F-GFP clone 5 cells incubated with talazoparib (blue), olaparib (purple), niraparib (red), veliparib (orange), 3-AB (gray), or iniparib (black) for 24 hours following DOX induction. Means from n = 2. e Summary of observed L713F-GFP stabilization and median DNA content EC₅₀ values for tested PARPi (in nM). f Concentration-dependent dynamics of PARP1 target engagement and DNA trapping in live cells after PARPi. Median GFP (y-axis) and Hoechst (x-axis) intensities are shown. Representative of n = 2, replotted from d. Light gray circle – –DOX control; dark gray circle – +DOX control; blue gradient circles – PARPi concentration gradient (3-AB – 12.8 nM to 1 mM; all other PARPi – 0.128 nM to 10 µM), red areas – PARP trapping phenotype. g Representative single cell, 2D plots comparing GFP intensity (y-axis) and Hoechst intensity (x-axis) following DMSO (gray) and PARPi treatment (orange; replotted from d). Inferred G1 and G2/M cell cycle phases demarcated by gray columns. Overview data in f representative of n = 500 cells per group; individual cell plots in g are n = 1000 cells per group. RFU – relative fluorescence units.