Fig. 5: PARP trapping is modulated by the PIAS4–RNF4–P97 axis.
From: The ubiquitin-dependent ATPase p97 removes cytotoxic trapped PARP1 from chromatin
a, Schematic of the trap–chase experiment. b, Trapped PARP1 is processed in a PIAS4-dependent manner. Trap–chase experiment in wild-type and PIAS4–/– HCT116 cells. After PARP1 trapping, cells were chased in talazoparib-containing media. Samples were collected at the indicated time points for chromatin fractionation and western blotting. c, Trapped PARP1 is processed in a RNF4-dependent manner. Trap–chase experiment in wild-type and RNF4–/– MCF7 cells as in b. b,c, Data represent two biological replicates. WB, western blot; and WT, wild type. d, Representative confocal microscopy images from a PARP1–γH2AX PLA trap–chase experiment. e, PARP1–γH2AX PLA foci persist in cells chased in PARPi plus p97 inhibitors. Number of PARP1–γH2AX PLA (anti-PARP1 + anti-γH2AX) foci in the trap–chase experiment in d; n = 5,736 cells from three independent experiments. f, PARP1–γH2AX PLA foci persist in cells with RNF4 silencing. Number of PLA foci in n = 1,235 cells from three independent experiments; siCon, control siRNA; and siRNF4, siRNA to RNF4. g, PARPi-induced RAD51 and γH2AX foci persist in the presence of p97 inhibitors. Representative confocal microscopy images from a trap–chase experiment (trap, talazoparib overnight; chase, p97 inhibitor-containing media) are shown for each condition. The cells were stained for the presence of γH2AX and RAD51 foci. Black box represents cells that were chased after talazoparib treatment, in contrast to the last two columns, which were not pre-treated with talazoparib. d,g, Scale bars, 5 μm. DMSO, dimethylsulfoxide. h,i, Number of γH2AX (h) and RAD51 foci (i) from the experiment in g; n = 1,750 cells from three independent experiments. e,f,h,i, The geometric mean and 95% CI are shown. ****P < 0.0001; NS, not significant; ordinary one-way ANOVA.
