Sp8 lightning confocal microscopy

Nuclear accumulation of STAT1 following 60 min of IFNγ stimulation was determined by immunofluorescence. Cells were seeded in a 24-well plate containing coverslips at 2 × 10⁵ cells/well. The next day, following stimulation with IFNγ as noted above, cells were fixed and permeabilized using ice-cold methanol and kept at −20 °C for 20 min. Cells were subsequently thoroughly washed with PBS, and kept in blocking solution (5% FBS in PBST) for an hour. Cells were then incubated overnight at 4 °C in primary anti-STAT1 antibody (1:200; Clone D1K9Y; Cell Signaling 14994). The following day, cells were thoroughly washed in PBST and incubated in secondary antibody (1:1000, Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 555; Thermo Fisher Scientific A-21428) for an hour at room temperature. Nuclear staining was done using DAPI (1:1000; Thermo Fisher Scientific 62248). Cells were finally thoroughly washed, dried, mounted on slides, and imaged using Leica SP8 Lightning Confocal Microscopy. Acquired images were analyzed using ImageJ version 2.1.0/1.53c. For each group, fluorescence from 10 nuclei was quantified.

Yeast cells of WT, SUSY7/ura3, and positive transformants were cultured in YP liquid to mid log phase. After being collected by centrifugation, the cells were resuspended in phosphate buffer (25 mM Na₂HPO₄, pH = 4) with 1 mM esculin. Subsequently, the cells were cultured in a shaker at 30°C for 1 h, and then washed three times with PBS (pH = 7.4). Finally, the cells were observed by a confocal laser scanning microscope (Leica SP8 lightning confocal microscopy) using excitation wavelengths of 420–460 nm.