Extended Data Fig. 5: cGAS interacts with importin-α and translocates to the nucleus in a manner dependent on importin-α. | Nature

Extended Data Fig. 5: cGAS interacts with importin-α and translocates to the nucleus in a manner dependent on importin-α.

From: Nuclear cGAS suppresses DNA repair and promotes tumorigenesis

Extended Data Fig. 5

a, Diagram showing the location and sequence of two predicted nuclear localization sequences of cGAS. b, Alignment of primary sequences of H. sapiens cGAS and its homologues in 22 species were performed as indicated in Extended Data Fig. 4a. The conserved NLS2 sequences are shown in the rectangular box. c, Immunoblot results of cell lysates and anti-Flag immunoprecipitates from HEK293T cells that had been transfected with HA–cGAS and the indicated Flag–KPNA and then exposed to etoposide (100 μg ml−1) for 4 h. Data represent n = 3 independent experiments. d, Immunoblot findings of cell lysates or anti-Flag immunoprecipitates from HEK293T cells that had been transfected with Flag–KPNA2 and HA–cGAS or the cGAS nuclear localization sequence deletion mutants (HA–cGAS(ΔNLS1) and HA–cGAS(ΔNLS2)) and exposed to etoposide (100 μg ml−1) for 4 h. Data represent n = 2 independent experiments. ej, Immunofluorescence findings showing the localization of transfected HA–cGAS or HA–cGAS(ΔNLS2) (anti-HA, green) in PC-9 cells treated with etoposide (100 μg ml−1) (e) or camptothecin (1 μM) (g) for 4 h or with H2O2 (10 mM) for 30 min (i). Nuclei were stained with DAPI (blue). Data represent n = 3 independent experiments. Quantitative data for e, g, i are shown in f, h, j, respectively, and are expressed as mean ± s.e.m. of n = 3 independent experiments. kp, 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) (m) for 4 h or with H2O2 (10 mM) for 30 min (o) in the presence of dimethyl sulfoxide (DMSO; mock treatment) or importazole (1 μM). Nuclei were stained with DAPI (blue). Data represent n = 3 independent experiments. Quantitative data for k, m, o are shown in l, n, p, respectively, and are expressed as mean ± s.e.m. of n = 3 independent experiments. At least 100 transfected cells were counted in each experiment. Student’s t tests (unpaired and two-tailed) were used for statistical analysis (f, h, j, l, n, p). Scale bar, 5 μm. For gel source data, see Supplementary Fig. 1.

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