Extended Data Fig. 4: OASL liquid phase separation during virus-induced necroptosis.
a, (Left) Representative images of liquid droplet formation of GFP-tagged full-length OASL, N-terminus OAS-like domain, or C-terminus UBL domain with mCherry-tagged RIPK3295-518. Scale bar, 5 μm. (Right) Histogram of the size and formation frequency of RIPK3295-518-mCherry droplets with or without the presence of GFP-tagged OASL proteins. b, (Left) Full length RIPK3-mCherry undergoes phase separation in the presence of purified OASL or N-OAS, but not C-UBL. Representative images of liquid droplet formation of mCherry-tagged full-length RIPK3 in the presence of full-length OASL, N-OAS or C-UBL. 2 µM of RIPK3-mCherry was mixed with 0.5 μM of purified OASL proteins. Scale bar, 5 μm. (Right) Histogram of the size and formation frequency of RIPK3 droplets. c, Confocal imaging of mock-infected Oasl1-/- primary fibroblasts reconstituted with mCherry-tagged vector, OASL1, or OASL1RK. Scale bar, 10 μm. d, (Top) OASL1-mCherry merged image of Fig. 4c with line across the condensate. Scale bar, 10 μm. (Bottom) Line profile of fluorescence intensity indicates colocalization of OASL1, ZBP1, and RIPK3 signal in OASL1-mCherry expressing Oasl1-/- primary fibroblasts. e, Quantitative analysis of morphological (top) and biochemical (bottom) parameters of OASL-GFP alone, RIPK3295-518-mCherry alone, and OASL-GFP with RIPK3295-518-Cherry liquid-like droplets before and after incubation at physiological conditions (n = 13 condensates per group). Morphological parameters include volumes, surface areas, and sphericity. Biochemical parameters include dry mass, concentration, and mean RI. ND, not detected. Data are representative of three (a-c) or two (e) independent experiments with similar results.
