Extended Data Fig. 6: cGAS interacts with γH2AX directly. | Nature

Extended Data Fig. 6: cGAS interacts with γH2AX directly.

From: Nuclear cGAS suppresses DNA repair and promotes tumorigenesis

Extended Data Fig. 6

a, b, Immunoblot of cell lysates and anti-H2AX (a) or anti-γH2AX (b) immunoprecipitates from PC-9 cells treated with etoposide (100 μg ml−1) for the indicated times. Data represent n = 3 independent experiments. c, Immunoblot of cell lysates or anti-HA immunoprecipitates from HEK293T cells transfected with Flag–cGAS and HA–H2AX in the absence or presence of etoposide (100 μg ml−1) for 4 h or H2O2 (10 mM) for 30 min. Data represent n = 3 independent experiments. d, e, Immunoblot of cell lysates or anti-Flag immunoprecipitates from HEK293T cells that had been transfected with Flag–H2AX and the indicated HA–cGAS truncated mutants in the presence of etoposide (100 μg ml−1) for 4 h. Data represent n = 3 independent experiments. f, Immunoblot of cell lysates or anti-Flag immunoprecipitates from HEK293T cells transfected with Flag–cGAS, HA–H2AX, or HA–H2AX(S139A). Data represent n = 3 independent experiments. g, Coomassie blue staining of purified GST–cGAS protein. Data represent n = 2 independent experiments. h, Binding curves of surface-immobilized nonphosphorylated peptide with GST-labelled cGAS at concentrations of 368 nM, 184 nM and 92 nM, and control GST protein at a concentration of 92 nM. Vertical lines mark the starts of association and dissociation phases of binding events. Data represent n = 4 independent experiments. i, Immunoblot of cell lysates of U2OS cells that had been stably transfected with either control shRNA or shRNA targeting H2AX (sh-H2AX). Data represent n = 2 independent experiments. j, k, Representative images of U2OS cells that had been stably transfected with either control shRNA or shRNA targeting H2AX, showing the recruitment of GFP–cGAS to sites of DNA damage induced by laser microirradiation. Data represent n = 3 independent experiments. Quantification data are shown in k and represent the mean ± s.d. of n = 17 (sh-Ctrl) and n = 22 (sh-H2AX) cells, respectively, from 3 independent experiments. Two-way ANOVA was used for statistical analysis. For gel source data, see Supplementary Fig. 1.