Fig. 3: SS18 undergoes liquid–liquid phase separation (LLPS) in vitro and in vivo.

a Analysis of SS18 387 amino acid protein sequence. Known domains SNH and QPGY. Red line predicts intrinsically unstructured regions (IUPred) score for intrinsically disordered tendencies; >0.5 is considered disordered. b Fluorescence and bright-field images of the SS18 droplets at varying protein concentrations. 1,6-hexanediol (Hex, 5%) was added to the SS18 protein (60 µM) to disrupt droplet formation. Liquid droplets are enriched in Alexa Fluor 488-labeled SS18 (1:100 molar ratio of labeled to unlabeled SS18). This protein labeling ratio was used throughout the study unless otherwise stated. The scale bar indicates 10 μm. c The small droplets underwent time-dependent dynamic fusion in the buffer comprised 50 mM Tris-HCl pH 7.5 and 150 mM NaCl at room temperature. Representative SDS-PAGE analysis (d) and quantification data e showing the distribution of proteins between aqueous solution/supernatant (S) and condensed liquid droplets/pellet (P) fractions for SS18 protein (30 µM) at different temperatures. The band intensities of proteins were quantified with Image J v1.8.0 software. Quantitative data represent results from three independent batches of sedimentation experiments and are plotted as mean ± SEM. f Live-cell imaging (GFP) and concurrent phase-contrast imaging for GFP-SS18 and GFP-SNF11 in HEK293T cells. The scale bar indicates 5 μm. g Representative time-lapse FRAP images showing that GFP-SS18 signal within the puncta recovered within a few seconds in HeLa cells. Red boxes show the zoomed-in regions. h FRAP recovery curves for six GFP-SS18 puncta from independent six HeLa cells with error bars indicating mean ± SEM. Time 0 refers to the time point of the photobleaching pulse.