Plan apo 1.4 na oil objective

Two days prior imaging cells were transiently transfected with plasmid encoding eGFP-53BP1 which was a gift from Daniel Durocher (Addgene plasmid #60813; http://n2t.net/addgene:60813; RRID:Addgene_60813, Watertown, MA, USA). Live cell imaging was performed at 37 °C and 5% CO₂ in a humidified stage top incubator (Tokai Hit, Shizuoka, Japan). Nikon Ti-E microscope with 60× Plan Apo oil objective (NA 1.4) and Nikon C2plus camera was used to collect multipoint time-lapse live cell images for 1 h with a temporal resolution of 5 min/frame. Imaging conditions were adjusted to minimize phototoxicity.
FRAP experiments were performed on the Nikon Ti-E microscope with 60× Plan Apo oil objective (NA 1.4) and Nikon C2plus camera at 37 °C in a humidified incubator. For bleaching and imaging of eGFP-53PB1, a 488-nm laser was used. After bleaching cells were imaged every 5 s for 5 min. Resulted images were analyzed using ImageJ software (version 1.44).

HeLa cells were seeded in 24-well plates on coverslips and transfected with plasmids coding for GR₂-GFP-NOSIP or Rev-GR-GFP. Import of GFP-reporter proteins was induced by adding 5 μM dexamethasone (Calbiochem) in DMEM containing 100 μg/μl cycloheximide for 1 h at 37 °C. Cells were washed three times with PBS and fixed in 3.7% formaldehyde for 10 min. For analysis of nuclear export, import was induced as described above. After three washing steps with PBS, cells were incubated with fresh DMEM containing cycloheximide (100 μg/μl) and lacking dexamethasone for 2 h at 37 °C, washed again 3 times with PBS, fixed in 3.7% formaldehyde in PBS for 10 min, and mounted in Mowiol containing DAPI. Cells were analyzed by fluorescence microscopy using a Nikon Ti-2 eclipse with a 60× Plan Apo 1.4 NA oil objective.