Extended Data Fig. 7: PARP1 trapping is modulated by PIAS4, RNF4 and p97.

a. Quantification of the chromatin bound PARP1 in Fig. 5b; 2 biological replicas are displayed with individual points. b. Quantification of the chromatin bound PARP1 in Fig. 5c; 2 biological replicas are displayed with individual points. c. As described in Fig. 5a, trapping was induced in cells and subsequently chased as stated. Cells were then fractionated and the amount of chromatin-bound PARP1 was investigated by Western blotting. Data shown represent 3 biological replicas. d. CAL51 PARP1^WT-eGFP cells were transfected with FLAG-RNF4-WT or FLAG-RNF-M136S/R177A (E2 binding mutant, dominant negative) constructs. Treatment occurred 24 h after expression. Data shown represent 2 biological replicas. e. HeLa cells were transfected with either p97-Strep-MYC cDNA or a p97 E578Q mutant-Strep-MYC. Sixteen hours later, cells were exposed to MMS + talazoparib. Data shown represent 2 biological replicas. f. CAL51 cells expressing PARP1^WT-eGFP or PARP1^{del.p119K120S}-eGFP were transfected with p97^WT-Strep-MYC or p97^E578Q-Strep-MYC-expressing construct. After trapping and pre-extraction, cells were fixed and imaged. Scale bar = 5 µm. g. Graph of quantification of PARP1–eGFP foci of the experiment presented in (F). n = 80-200 cells examined over 3 biologically independent experiments, mean ± SEM, p-values derived with a Kruskal-Wallis test. h. Western blots of trapped PARP1 from CAL51 PARP1–eGFP expressing cells transfected with a control siRNA (siLuc) or UFD1-targeting siRNA (siUFD1). 72 hours post transfection, cells were treated with MMS + talazoparib. Data shown represent 3 biological replicas.