Fig. 4: XBP1s undergoes phase separation in vitro and form condensates in the nucleus.

A Domain structure and graphs of XBP1s IDRs based on VSL2 and IUPred algorithms. Scores >0.5 indicate disorder. Yellow shade depicts the designated core IDR (cIDR). B Representative fluorescence images (488 nm) of XBP1s droplets at varying protein concentrations (5 µM–30 µM) in a buffer containing 150 nM NaCl and 20% PEG-8000 (default condition unless otherwise specified). Both the size and number of droplets increased as protein concentration rose. Quantification of droplet area is shown on the right. C Representative fluorescence images (488 nm) of XBP1s-droplets at different temperatures. The droplet number peaked at 37 °C. Quantification of droplet area is shown on the right. D, E Representative fluorescence images (488 nm) of XBP1s-droplets at different NaCl concentrations (25–1000 nm). At 150 mM NaCl, droplet numbers were the highest, while at 200 mM NaCl, droplets were larger but fewer, indicating a concentration-dependent phase separation behavior. F Fluorescence recovery after photobleaching (FRAP) analysis of EGFP-XBP1s droplets in vitro. The graph illustrates the recovery of fluorescence intensity within 0–60 s, indicating the liquid-like dynamics of the droplets. G Live-cell imaging of A549 and PC9 cells, EGFP-XBP1s puncta were observed under treatment with buffers, both in the presence and absence of 5% 1,6-hexanediol. Nuclei were distinctly visualized through Hoechst staining. H Live-cell imaging showed the fusion phenomenon of XBP1s droplets was observed through live-cell imaging in A549 and PC9 cells, indicating the liquid-like dynamics of the droplets. I FRAP recovery of EGFP-XBP1s puncta in live A549 and PC9 cells. White squares mark photo-bleached puncta. The fluorescence recovery occurred within 2–30 s in A549 cells and 2–38 s in PC9 cells, further confirming the dynamic and liquid-like properties of XBP1s condensates. Quantification of fluorescence recovery is displayed on the right.