Axioplan 2 upright fluorescence microscope

GFP-tagged wild-type (WT) and mutant WDR11 expression constructs were transfected into HEK293T. After 48 hours, cells were treated with leptomycin B (10 ng/mL) for 10 hours before being fixed with 4% paraformaldehyde. After being washed three times in phosphate-buffered saline, the nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) and the cover slips were mounted in Mowiol 4-88 (Fluka; Sigma-Aldrich). Images were analyzed by using Zeiss Axioplan 2 Upright fluorescence microscope (Carl Zeiss, Oberkochen, Germany) and ImageJ software (National Institutes of Health, Bethesda, MD). In each experiment, approximately 200 cells were scored for the nuclear or cytoplasmic location of WDR11 based on the GFP signal at 488 nm, against the total cells in the field based on the DAPI signal at 405 nm.

Immunofluorescence experiments were performed as previously described [44 (link)]. At least 100 cells were analyzed per experimental group.
PLA was conducted according to manufacturer’s instructions (Duolink kit^®, Sigma-Aldrich). Cells were incubated with a pair of anti-Cyclin B1 and anti-Cdk1antibody, followed by secondary proximity probes (PLA probe PLUS and MINUS). Subsequently, they were incubated with the ligation solution containing ligase and amplification solution containing polymerase and a fluorophore with 594nm excitation and 624 nm emission. Then, cells were incubated with anti-β tubulin antibody and appropriate secondary antibody. Fluorescence signals were detected by Zeiss Axioplan 2 upright fluorescence microscope (40X objective; Carl Zeiss) and analyzed with AxioVision software (Carl Zeiss). PLA-positive signals were quantified using Duolink Image Tool (Sigma-Aldrich). At least 30 cells were analyzed per experimental group.