Fig. 2: LLPS ability of SARS2-NP can be enhanced by its poly-SUMOylation.

MST assaybetween ligand EGFP-SARS2-NP (a) or Cy5-RNA (b) and SARS2-NP or SUMO3-SARS2-NP. Data points indicate the difference in normalized fluorescence (Fnorm, ‰). c Schematic of SUMO3-SARS2-NP chimeric protein with a TEV protease cut site in-between (left). The cleavage result was analyzed by Coomassie-stained SDS-PAGE (right). d Droplet formation of AF488-labeled SARS2-NP WT, or SUMO3-SARS2-NP (without or with cleavage of TEV protease) without or with Cy5-RNA. Left, representative images. Right, fold change in droplet formation. e Fusion of AF488-labeled SARS2-NP WT, or SUMO3-SARS2-NP droplets without or with Cy5-RNA, at pH 5.5, 150 mM (left). And equivalent diameter (EqDiameter) frequency distribution of liquid droplets formed at 30 s (right). f FRAP assay of AF488-labeled SARS2-NP WT, or SUMO3-SARS2-NP droplets without or with Cy5-RNA. Left, representative images of before and after photobleaching. Right, quantification of FRAP over a 30 s time course. MST assay between ligand AF488-labeled SARS2-NP (g) or Cy5-RNA (h) and HEK293F cells-purified SARS2-NP WT/K65R. i Droplet formation of AF488-labeled SARS2-NP WT /K65R purified from HEK293F cells, without or with Cy5-RNA. Left, representative images. Right, fold change in droplet formation. j Puncta formation of EGFP-SARS2-NP WT/K65R in HeLa cells. Left, representative fluorescence images. Nuclei were visualized using DAPI staining. Middle, the percentages of cells harboring puncta in fluorescence positive cells. Right, the ratio of puncta-like fluorescence intensity. All data are representative of at least three independent experiments with similar results. Data are presented as Mean  ±  SD (a, b, d, f–j). n = 3 (a, b, f, g, h), or 6 (d, i, j middle), or 10 (j right) independent samples. Statistical analyses were performed using a One-way ANOVA (d, i, j), or Two-way ANOVA (a, b, f–h). Scale bar, 10 μm (d–f, i, j). DIC, differential interference contrast (d, i).