Fig 2: IDR1 is required for RAP80 phase separation. (A) IDRs of RAP80 were analyzed using PONDR (http://www.pondr.com/) and the diagram of RAP80 mutants. (B) living cell imaging of IDR1-mEGFP, IDR2-mEGFP, IDR3-mEGFP and ΔIDR1-mEGFP in HEK 293T cells. Twenty-four hours after plasmid transfection, GFP-positive cells were selected using FACS and seeded in plates for living cell imaging and western blotting analysis. The foci number of all cells observed in living cell imaging was counted. Scale bar, 5 μm. (C) FRAP assay on IDR1-mEGFP condensate in HEK 293T cells. Scale bar, 5 μm. (D) Confocal image of in vitro condensates formation of GST-RAP80-mCherry, GST-IDR1-mCherry, and GST-ΔIDR1-mCherry protein. (E) OptoIDR assay to verify the IDR1-mediated LLPS. n = 6. Scale bar, 5 μm. (F) FRAP assay of blue light-induced IDR1-mCherry-CRY2 droplets. Blue light, 60 s. Scale bar, 5 μm.

Fig 3: Lys63-linked poly-ubiquitin triggers RAP80 phase separation in a UIMs- and IDR1-dependent manner. (A) 1.2 μM GST-RAP80-mCherry was incubated with 0.6 μM Lys63-linked poly-ubiquitin containing the indicated number of units. The fluorescence intensity of droplets was presented as the area × mean intensity (A × M). Scale bar, 2 μm. (B) Long Lys63-linked poly-ubiquitin induced RAP80 LLPS stronger than a short one. The fluorescence intensity of droplets was presented as the area × mean intensity (A × M). Scale bar, 2 μm. (C) Diagram of the RAP80 protein-truncating mutants. (D) Lys63-linked poly-ubiquitin trigger RAP80 LLPS in a UIM-depended manner. The fluorescence intensity of droplets was presented as the area × mean intensity (A × M). Scale bar 2 μm. (E) examining the affinity of GST-RAP80-mCherry, GST-IDR1-mCherry, and GST-(SIM+UIM)-mCherry to poly-ubiquitin using GST pull-down assay. (F) Long K63 poly-ubiquitin chains triggered RAP80 IDR1 LLPS. The fluorescence intensity of droplets was presented as the area × mean intensity (A × M). Scale bar, 2 μm.

Fig 4: RAP80 siRNA transfection decreased the invasive or migrating ability of MCF-7 breast cancer cells.Notes: Transwell migration assay shows the migrating ability of wild-type MCF-7 (A), MCF-7-siNC (B), and MCF-7 siRAP80 (C) at 72 hours after transfection. (G) Number of cells migrating onto the lower surfaces of the filter was counted. The transwell migration assay shows the invasive ability of wild-type MCF-7 (D), MCF-7-siNC (E), and MCF-7 siRAP80 (F) at 72 hours after transfection (H). Number of cells invading onto the lower surfaces of the filter was counted. Data are presented as mean ± SD of three independent experiments, *P<0.05 vs MCF-7 siRAP80 group.Abbreviation: NC, normal control.

Fig 5: The Effect of RAP80 siRNA transfection on cell proliferation by MTT in wild-type MCF-7 or MCF-7 RAP80 siRNA.Notes: The survival rate of both cells decreased in a dose-dependent manner and the IC50 value for cisplatin in MCF-7 RAP80 siRNA cells was 0.83 µg/mL, and 1.69 µg/mL in wild-type MCF-7. Cell viability was measured by MTT; n=3 for each data point.