SARS-CoV-2 nucleocapsid protein undergoes liquid–liquid phase separation into stress granules through its N-terminal intrinsically disordered region

Materials and Methods Plasmid constructs The cDNA encoding N protein was cloned into pEGFPN1 plasmid using the standard polymerase chain reaction (PCR) and restriction enzymes method. Truncations and deletions of N plasmid were made using similar method with appropriate primers. DNA fragments encoding N protein were inserted into pGEX-6P-1 plasmid using pEASY-Uni Seamless Cloning and Assembly Kit (Transgen, CU101) for purification of N protein. The pEGFP-C1-G3BP1 plasmid (#135997) was purchased from Addgene. DNA fragments encoding mCherry and G3BP1 were amplified using mCherry-N1 plasmid and pEGFP-C1G3BP1 plasmid separately, then they were assembled and inserted into pCDNA3.1(-) plasmid by pEASY-Uni Seamless Cloning and Assembly Kit.

PBS for 45 min, then washed three times with PBS, followed by incubation with G3BP1 antibody (CST, #61559) diluted in incubation buffer (1% BSA, 0.02% Triton X-100 in PBS) for one hour at room temperature. After wash three times with PBS, the samples were incubated with Alexa Fluor 568 conjugated secondary antibody (Thermo Fisher, #A11011) for one hour respectively, then stained with DAPI (0.1µg/ml) in PBS and sealed with mounting buffer. Images were collected using a Nikon A1R MP confocal microscope under an 100× oil-immersion objective. All images were processed with Image J software.

FRAP and live cell imaging
HeLa Cells were transfected with indicated plasmids and grown on a culture dish with glass bottom for 20-24 h. Cells were transferred to a living cell station (Oko Lab) and cultured at 37°C, 5% CO 2 , and 80% humidity. Arsenite was added 30 min before observation under Nikon A1R MP confocal microscope. For FRAP experiment, the green puncta of appropriate size were chosen and bleached by 0.25 s with 100% laser power (488 solid state laser), whereafter scanned with a 30 s interval of 3 min in total. Mean optical intensity of the bleached puncta were calculated applying Image J software. All collected data were analyzed with Excel 2013 software and graphed in Graphpad Prism 5 software. For live cell imaging, the cells with droplets were photographed at 30 s interval in a duration of 1 hour. All images were processed with Image J software.

Statistics of puncta
The criteria we used to define the ratio of cells with puncta were as follows: The number of N-GFP expressing cells bearing more than one puncta with a diameter of more than one micrometer was calculated as a. The number of N-GFP expressing cells without puncta was calculated as b. The percentage of cells with puncta equaled a/(a+b).
The number and area of puncta in cells were automatically calculated using ImageJ software and analyzed by Excel software.

Liquid-liquid phase separation
The in vitro LLPS experiments were conducted at room temperature. Proteins were mixed at a desired concentration, and the concentration of NaCl was adjusted with 5 M NaCl and 25 mM HEPES (pH 7.5). HeLa cell total RNAs were extracted with RNAsimple Total RNA Kit (TIANGEN, DP419). The concentration of RNAs were quantified by Nanodrop (Thermo Fisher). Protein solutions were prepared 15-30 min before imaging at room temperature.For imaging, protein solutions were loaded onto a glass-bottom 384 well plate.
Images were taken by Nikon A1R MP confocal microscope using a 100× oil-immersion objective.