Fig. 5: LLPS of 53BP1 and HP1α.

a Generation of GFP-53BP1 fragments. b Purified proteins were mixed on ice, incubated at room temperature for 20 min, centrifuged at 10,000 g for 10 min and visualized by the Tanon 2500 Imager at 590 nm. Far Right: short exposure for the mixture of HP1α and GFP. Red circles indicate precipitations after centrifugation. c Representative bright field and single z-plane confocal images of liquid droplets formed by mGFP-53BP1-C1, -C2 or -C3 with mCherry-HP1α in vitro. Lower panel: smaller droplets. Scale bar is 40 μm. d MCF-10A parental or 53BP1 KO cells were transfected with GFP-HP1α for 48 h and FRAP was performed. Representative single z-plane confocal images are shown. Right: protein expression. The residual 53BP1 signal in the KO lane might have come from loading contamination. e FRAP analysis of cells in (d) from n = 14 and 24 events. f U-2 OS parental or HP1α KD cells were transfected with GFP-53BP1-C1 for 48 h and FRAP was performed. Representative single z-plane confocal images are shown. Right: protein expression. The arrow indicates a 53BP1-C1 punctum that transiently recovered (60 s) but then disappeared (120 s). g FRAP analysis of cells in (f) from n = 20 and 18 events. The X-axis (time) in (e) and (f) is not scaled and 0 indicates photobleaching. Red circles in (d) and (f) indicate puncta that were bleached. Scale bars in (e) and (f) are 10 μm. FRAP quantitation in (e) and (g) represent mean values and SD acquired from two independent experiments.