Fig. 2. CTCF exhibits phase separation behavior in the nucleus.

a Mouse CTCF is an intrinsically disordered protein predicted by IUPred2A;⁸⁸ the score > 0.5 indicates IDR. b SIM microscopic images of CTCF immunofluorescence and live cell imaging in ESCs. Live imaging of ESCs expressing an EGFP-tagged CTCF.¹⁶ c Representative images (left) and quantification (right) of FRAP in ESCs expressing exogenous CTCF-EGFP. The yellow box highlights the bleached puncta. Data are plotted as means ± SEM, n = 3. d Droplet fusion and fission behavior of CTCF puncta in CTCF-EGFP ESCs. e Relative number of different volumes of CTCF puncta upon 1.5% 1,6-hex treatment. Welch’s t-test; vehicle, n = 60 cells; 1,6-hex, n = 82 cells. P values are (from left to right): P = 0.0045, P = 0.0632, P = 0.8989, P = 0.3059. f Representative images of droplet formation at different concentrations of NaCl and proteins. g Representative images of droplet formation at 1 µM, 5 µM and 10 µM CTCF in the presence of 20% PEG8000. h Representative images of CTCF-mCherry aggregation after addition of Cy5-labbled 25 × DNA motif. The concentration of CTCF-mCherry was 0.8 µM. n.s., not significant, P > 0.05; **P < 0.01.