Fig. 3: RDM16 undergoes condensation in vitro.
a In vitro phase separation assays of 10 μM GFP or GFP-RDM16 in the presence of 10% (w/v) PEG4000. Scale bars, 5 μm. b In vitro phase separation assay of 10 μM GFP or GFP-RDM16 in the presence of 15% (w/v) Ficoll 400. Scale bars, 5 μm. c Phase separation assay of purified GFP-RDM16 at different concentrations in the presence of 10% (w/v) PEG4000. Scale bars, 5 μm. d In vitro phase separation assay of 10 μM GFP or GFP-RDM16 in the presence of 10% (w/v) PEG4000. The addition of 15% (w/v) 1,6-hexanediol solution disrupted RDM16 phase separation. Scale bars, 5 μm. e GFP-RDM16 phase separation assay with different concentrations of protein and NaCl. Scale bars, 5 μm. f Fusion of GFP-RDM16 liquid droplets. Scale bars, 2 μm. g FRAP of GFP-RDM16 droplets (10 μM concentration) in the presence of 10% (w/v) PEG4000. Time 0 s indicates the time of the photobleaching pulse. Scale bars, 1 μm. h Recovery of GFP-RDM16 fluorescence after photobleaching. Data are means ± SD, n = 6. i Effect of temperature treatment on 10 μM GFP-RDM16 droplets in HEPES buffer with and without 10% (w/v) PEG4000. GFP-RDM16 droplets increase as the temperature rises from 22 °C to 37 °C or 45 °C. Scale bars, 10 μm. j, k Droplet size (j) and fluorescence intensity (k) of GFP-RDM16 shown in (i). n = 199, 240, 219, 216, and 217 for 22 °C, 37 °C, recovery (37 °C–22 °C), 45 °C, and recovery (45 °C–22 °C). l Images and quantification of FRAP of GFP-RDM16 droplets after photobleaching under the condition of HS at 37 °C. Scale bars, 2 μm. Data are means ± SD, n = 6. The maximum recovery fraction (Rmax) and half-time of recovery (t1/2) in (h, l) was calculated from the logarithmic curve and the best-fit values were generated by GraphPad. In (j, k), different letters indicate significant differences by non-parametric version of one-way ANOVA: Kruskal–Wallis test followed by Dunn’s multiple comparisons test (P < 0.05). All experiments were performed at least twice with similar results.
