Fig. 3: OASL undergoes LLPS during virus-induced necroptosis.
a, Fluorescence images of droplet formation by GFP-tagged full-length OASL, and the N-OAS and C-UBL domains of OASL in vitro 30 min after induction. b, LLPS of GFP-tagged OASL over time in vitro. c, Time-lapse images of the coalescence of OASL liquid droplets over 120 s (indicated by the arrow). d, Turbidity measurement of GFP-tagged full-length OASL, and the N-OAS and C-UBL domains of OASL over time in vitro. e, Representative 3D RI distribution and fluorescence of OASL droplets before and after incubation at physiological conditions (green, GFP fluorescence; purple, RI tomogram; n = 50 condensates per condition). f, Statistical analyses of morphological (top) and biochemical (bottom) parameters of e (n = 7 condensates per condition). ND, not detected. g, Representative images of OASL–GFP (0.05 μM) phase separation mixed with poly(I:C) LMW (50 μg ml–1), poly(I:C) HMW (50 μg ml–1) or RNase A (300 μg ml–1) for 60 min. h, Droplet formation of full-length OASL–GFP and the dsRNA-binding mutant OASL-RK–GFP. i, Representative images of Oasl1–/– primary fibroblasts reconstituted with OASL1–mCherry (OASL1–mCh) and infected with MCMV-M45mutRHIM. Viral dsRNA was immunostained with J2 antibody. j, Representative images of Oasl1–/– primary fibroblasts reconstituted with HA–vector, HA–OASL1 or HA–OASL-RK and infected with MCMV-M45mutRHIM. Arrows indicate discrete OASL1 foci in the cytosol. k, Top: arrows indicate the SYTO-45-stained OASL1–mCherry area chosen for photobleaching. Bottom: representative images of OASL1–mCherry foci before and after photobleaching (n = 3 cells). Rectangle frames represent the bleached and recovered area within the targeted area. l, Quantitative FRAP of OASL1 and SYTO 45 foci for 160 s in Oasl1–/– fibroblasts reconstituted with OASL1–mCherry and infected with MCMV-M45mutRHIM for 4, 6 or 8 h. Calculated exponential constant (K) and normalized plateau after fluorescence recovery (R) are the mean ± s.e.m. (n = 2 OASL1 and SYTO 45 foci). Images are representative of five (a–c,g,h), three (d–f,i,j) or two (k,l) independent experiments. For f, data are presented as the mean ± s.e.m. and statistical analysis was performed using two-tailed unpaired t-test. Scale bars, 10 μm (a–c,g,i–k) or 30 μm (h).
