Extended Data Fig. 9: cGAS interacts with PARP1 via PAR and prevents formation of the PARP1–Timeless complex. | Nature

Extended Data Fig. 9: cGAS interacts with PARP1 via PAR and prevents formation of the PARP1–Timeless complex.

From: Nuclear cGAS suppresses DNA repair and promotes tumorigenesis

Extended Data Fig. 9

a, Coomassie-blue-stained SDS-polyacrylamide gel image showing the anti-HA immunoprecipitates of HEK293T cells stably transfected with pcDNA3.1-HA (HA) or HA–cGAS (HA–cGAS) and treated with H2O2 (10 mM) for 30 min. M, pre-stained protein markers. Distinct protein bands (indicated as 1 and 2) in the anti-HA immunoprecipitates of HEK293T cells that had been stably transfected with HA–cGAS were identified by mass spectrometry. Data represent n = 2 independent experiments. b, Results of mass spectrometry analysis of bands 1 and 2 in the anti-HA immunoprecipitates of HEK293T cells that had been stably transfected with HA–cGAS. c, Immunoblot of cell lysates or anti-Flag immunoprecipitates from HEK293T cells that were transfected with Flag–PARP1 and HA–cGAS in the presence or absence of DNase I. Data represent n = 3 independent experiments. d, Immunoblot of cell lysates or anti-Flag immunoprecipitates from HEK293T cells that were transfected with Flag–cGAS and HA–PARP1 in the absence or presence of the PARP1 inhibitor, olaparib (20 μM). Data represent n = 3 independent experiments. e, Immunoblot results of cell lysates or anti-Flag immunoprecipitates from HEK293T cells that had been transfected with Flag–cGAS, HA–PARP1 or its enzyme-inactivated mutant HA–PARP1(E988K). Data represent n = 3 independent experiments. fh, Immunoblot of cell lysates or anti-Flag immunoprecipitates from HEK293T cells that had been transfected with Flag–PARP1, Myc–Timeless and HA–cGAS and then exposed to etoposide (100 μg ml−1) (f) or camptothecin (1 μM) (g) for 4 h, or ionizing irradiation (8 Gy) (h). Data represent n = 3 independent experiments. i, j, Representative images of U2OS cells showing the recruitment of GFP–PARP1 to sites of DNA damage induced by laser microirradiation. Cells had been transfected with pcDNA3.1-HA control (HA) or HA–cGAS. Quantification data are shown in j and represent the mean ± s.d. of n = 19 cells (both HA and HA–cGAS) from 3 independent experiments. Two-way ANOVA was used for statistical analysis. NS, not significant. km, Representative immunofluorescence of HA–cGAS (anti-HA, green) in PC-9 HA–cGAS cells treated with etoposide (100 μg ml−1) (k) or camptothecin (1 μM) (l) for 4 h in the presence of DMSO (mock treatment) or olaparib (20 μM). Nuclei were stained with DAPI (blue). Data represent n = 3 independent experiments. Quantitative data are shown in m. At least 100 transfected cells were counted in each experiment. Data are expressed as mean ± s.e.m. of n = 3 independent experiments (m). Student’s t tests (unpaired and two-tailed) were used for statistical analysis. Scale bar, 5 μm. For gel source data, see Supplementary Fig. 1.