Supplementary Figure 4: In vitro assembly of MEG-3 and PGL-3 condensates.

a Coomassie gels of purified MEG-3 and PGL-3 proteins (Methods). b Representative photomicrographs of MEG-3 and PGL-3 images from Fig. 4a. Images were uniformly processed to reveal the small MEG-3 condensates at the onset of co-assembly. Images were taken at indicated time points after initiation of condensate assembly. Scale bar is 5 µm. c Histograms of intensity distributions of MEG-3 and PGL-3 condensates individually or together after 30 mins of condensate formation. These results suggest that in the individual condensation experiments, most PGL-3 condensates, but only a fraction of MEG-3 condensates, are detected under our imaging conditions. d Graph showing fluorescence recovery after photobleaching (FRAP) of PGL-3 condensates assembled as in Fig. 4a and incubated for 30 min. The entire PGL-3 condensate was bleached. Intensity was measured every 3 s for 300 s before and after bleaching. Values were normalized to initial fluorescence intensity, corrected for photobleaching and plotted as an average from multiple experiments (n = 8). Error bars represent mean ± SD. Representative images from FRAP experiments are shown below the graph. Scale bar is 1 µm. e Graphs showing the Pearson’s correlation coefficient comparing the intensity of MEG-3 or PGL-3 in each condensate. MEG-3 and PGL-3 show increased co-localization over time. Each data point represents the mean ± SD of coefficients calculated for a total of 16 images from 4 experimental replicates. f Radial intensity profile plot of the MEG-3 and PGL-3 phases from the co-condensate shown in Fig. 4d (30 min in vitro assembly). Lines represent the relative maximum intensity along radii extending from the condensate in 360^°. The resulting plot shows the variation in intensity around the circumference of the condensate. Note that PGL-3 is evenly distributed around the condensate, while MEG-3 intensity varies. g Photomicrographs of selected frames from Supplemental video 4. Co-condensates were assembled as in Fig. 4a and incubated for 15 min. Scale bar is 1 µm. Co-condensates of MEG-3 and PGL-3 fuse. MEG-3 remains enriched on the surface of the PGL-3 phase after fusion. h Photomicrographs of selected frames from Supplemental video 5. Co-condensates were assembled as in Fig. 4a and incubated for 15 min. Arrows indicate the position of MEG-3 condensates as they move relative to each other on the surface of the PGL-3 condensate. Scale bar is 1 µm. i Photomicrographs of selected frames from Supplemental video 6. Co-condensates were assembled as in Fig. 4a and incubated for 15 min. A MEG-3 condensate on the surface of the PGL-3 condensate was FRAPed (panel 2). The photobleached region indicated by the arrow changes position during the recovery period consistent with movement of the MEG-3 phase. Scale bar is 1 µm.