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. 2016 Dec 3;5:e21337. doi: 10.7554/eLife.21337

Figure 4. Stimulation of MEG-3 phase separation by RNA.

(A) Bar graph showing the number of condensates formed by 0.5 μM MEG-3 or MEG-3IDR in the presence of increasing poly-U30. Values represent averages from three technical replicates. Error bars indicate S.E.M. (B) Photomicrographs of phase separation assay showing condensate formation of 5 μM full length MEG-3 incubated with poly-U RNA and/or MEX-5 as indicated.(C) Violin plots showing condensate size and number for each experiment represented in B. The height of the plot shows the area of condensates in μm2. The width of the plot correlates to the proportion of condensates of that size. Numbers inside each violin plot are the total number of condensates pooled from three technical replicates for each condition. An additional control is shown in Figure 4—figure supplement 2.

DOI: http://dx.doi.org/10.7554/eLife.21337.009

Figure 4.

Figure 4—figure supplement 1. Phase Separation assay titration.

Figure 4—figure supplement 1.

DIC images of MEG-3 solutions in the presence of increasing concentrations of protein and RNA. MEG-3 was diluted into phase separation buffer (25 mM HEPES, pH7.4, NaCl adjusted to a final concentration of 150 mM) at room temperature. The solution was transferred to a glass bottom dish and the dish surface was photographed using an inverted DIC microscope 10 min after the initial dilution (room temperature).
DOI: http://dx.doi.org/10.7554/eLife.21337.010
Figure 4—figure supplement 2. Phase Separation assay controls.

Figure 4—figure supplement 2.

(A) Same as above except 3’ fluorescein labeled poly-U RNA was included in the buffer. DIC images (left) show the MEG-3 condensates and fluorescence images (488 channel) show the 3’ fluorescein labeled poly-U RNA concentrated in the condensates. (B) Violin plots showing condensate size and number comparing MEG-3 alone and MEG-3 + MEX-5 (Additional data). As in Figure 4C, the height of the plot shows the area of condensates in μm2. The width of the plot correlates to the proportion of condensates of that size. Numbers inside each violin plot are the total number of condensates pooled from three technical replicates for each condition. Data for the MEG-3 alone sample are the same as in Figure 4C and shown here only for reference.
DOI: http://dx.doi.org/10.7554/eLife.21337.011