Quantifying FUS-induced Stress Granule Formation

Briefly, cells were fixed 24 or 48 h (when FUS is co-transfected with siXPO1) after transfection in 4% formaldehyde in phosphate buffered saline (PBS) and rinsed three times. XPO1 staining was performed as described previously (35 (link)). We blocked the cells in wash buffer (PBS, 0.1% Tween-20 and 2 mg/ml Heparin) supplemented with 3% donkey serum and 5% glycine for 1 h. Subsequently, cells were incubated with the rabbit anti-mouse CRM1 antibody (Novus Biologicals, Littleton, CO, USA; NBP2–16014), diluted 1:100, for 16 h at 4°C. After washing the cells four times with wash buffer, Alexa Fluor-647 conjugated secondary antibodies (Invitrogen) were applied to the cells for 3 h at RT. Secondary antibodies were diluted 1:1000 in wash buffer with 3% donkey serum. By confocal microscopy, we analyzed the localization of XPO1. In order to determine the recruitment of FUS into cytoplasmic granules, cells were analyzed using the CellInsight™ CX5 High Content Screening (HCS) Platform (ThermoFisher Scientific; Cat#CX51110). Nuclear and cytoplasmic intensities were analyzed with the HCS studio cell analysis software (ThermoFisher Scientific). Stress granules were induced by incubating the cells for 1 h with 0.5 mm NaAsO₂ (Sigma).