Fig. 3: RNA probe and STED imaging reveal the molecular mechanism of hollow co-condensate formation.
From: Deciphering the molecular mechanisms of FET fusion oncoprotein–DNA hollow co-condensates
a–c 5 μM SNAP-FUS-ERG first mixed with 0.6 μM ATTO425-labeled 25-bp 4 × GGAA dsDNA (a(i)), random dsDNA (b(i)), or no dsDNA (c). Next, 5 ng/μL 50-nt Cy5-labeled PolyU RNA was injected. a(ii) and b(ii) are normalized intensity profiles from a(i) and b(i). d Boxplot of the mean intensity of RNA inside the condensates in a(i), b(i) and c. The total number N examined over three-time in vitro droplet experiments. Exact P values are as follows: 4 × GGAA dsDNA vs. random dsDNA, P < 0.0001; 4 × GGAA dsDNA vs. no dsDNA, P < 0.0001; random dsDNA vs. no dsDNA, P = 0.0016. e Schematics of hollow co-condensate formation. f (i) Representative fluorescence heatmap of Stimulated Emission Depletion (STED) microscopy image for 5 μM SNAP-FUS-ERG mixed with 25 bp 4 × GGAA dsDNA: 0.6 μM dark dsDNA and 0.006 μM dsDNA labeled with Atto647N (100:1). (ii) Boxplot of the radial intensity distribution of dsDNA labeled with Atto647N within the hollow co-condensates. The total number N examined over three-time in vitro droplet experiments. g (i) Representative fluorescence heatmap of STED image for 4 mg/mL FUS-RGG3 (FUS No. 471-504) mixed with Poly-U RNA: 20,000 ng/μL dark RNA and 100 ng/μL RNA labeled with AlexaFluor 647 (200:1). (ii) Boxplot of the radial intensity distribution of RNA labeled with AlexaFluor 647 within the hollow co-condensates. The total number N examined over three-time in vitro droplet experiments. For the boxplot in d, f(ii) and g(ii), the red bar represents median. The bottom edge of the box represents 25th percentiles, and the top is 75th percentiles. Most extreme data points are covered by the whiskers except outliers. The ‘+’ symbol is used to represent the outliers. Statistical significance was analyzed using unpaired t test for two groups. P value: two-tailed; p value style: GP: 0.1234 (ns), 0.0332 (*), 0.0021 (**), 0.0002 (***), < 0.0001 (****). Confidence level: 95%. Scale bar: 1 μm in a(i), b(i), c; 0.5 μm in f(i); 2 μm in g(i). Source data are provided as a Source Data file.
