Sir dna probe

Cells were grown on 8-well glass-bottom dishes (LabTek). Two hours prior to imaging, SiR-DNA probe (1:2000, Spirochrome) was added together with verapamil (1:10000) to visualize DNA in L-15 CO₂-independent medium (GIBCO). Images were taken using a Deltavision deconvolution microscope (Applied Precision). Cells were imaged every 5 minutes using a 40x air objective with 3 × 3 μm Z stacks. Image acquisition was carried out using Softworx (Applied Precision) and ImageJ.

Cells were transfected with a pC1-EGFP-CAP-H construct in 4-well glass bottom dishes (LabTek). 48 hours post-transfection, SiR-DNA probe (1:2000, Spirochrome) was added to cells to visualize DNA, in L-15 CO₂-independent medium (GIBCO). FRAP experiments were performed using a confocal microscope (Leica Microsystems) with a 63x oil immersion objective using the LAS-AF FRAP-Wizard. Images were taken by accumulating 4 frames at a scanning speed of 1000 Hz. Photobleaching was performed by 6 times illuminating the selected region at 100-fold laser power (488 nm Laser). 60 post-bleaching images were taken with 10 s intervals. Analysis of images was done using an in-house ImageJ macro. A mask on the DNA signal was created and the EGFP-CAP-H signal was normalized to the DNA signal.