Fig. 6: BRRF2 inhibits cGAS phase separation in cells and in vitro.

A Left panel, Representative confocal images acquired from HEK293T/GFP-cGAS cells transiently expressing mCherry-BRRF2 or an empty vector, which were transfected with HT-DNA (2 μg/mL) for 12 h. The cells were fixed with 4% paraformaldehyde and stained with DAPI. Right panel, Histogram of the percentage of cells with GFP-cGAS foci. The data were collected from 100 cells. (The means ± SDs are shown. ****p < 0.0001 according to two-way ANOVA followed by Sidak’s multiple comparisons test). B Left panel, Representative confocal images acquired from HEK293T cells transiently expressing mCherry-BRRF2 or an empty vector, which were transfected with HT-DNA (2 μg/mL) for 12 h. The cells were fixed with 4% paraformaldehyde for immunofluorescence staining with the indicated anti-G3BP1 antibody. Right panel, Histogram of the percentage of cells with G3BP1 foci. The data were collected from 100 cells. (The means ± SDs are shown. ***p < 0.001, ****p < 0.0001 by two-way ANOVA followed by Sidak’s multiple comparisons test). C Diagram of the sedimentation assay used to separate the condensed liquid phase and the aqueous phase for immunoblot assays. D Separation of the condensed liquid phase and the aqueous phase of His-cGAS and HT-DNA. His-cGAS was mixed with or without HT-DNA for phase separation in the presence of GST or the indicated amount of GST-tagged BRRF2 for 1 h at 37 °C. The condensed liquid phase and the aqueous phase were prepared for immunoblot assays with the indicated antibodies. The experiment was performed as three independent biological replicates. Representative images was shown. Source data are provided as a Source Data file. E Representative DIC time-lapse imaging of Cy5-dsDNA and FITC-cGAS liquid droplets in the presence/absence of mCherry-BRRF2Δ322-573. The experiment was performed as three independent biological replicates.