Sir dna reagent

HeLa cells were treated with CellLight Tubulin-GFP and BacMam 2.0 (Thermo Fisher Scientific) for 12 h before siRNA transfection. Transfection of siRNAs into HeLa cells at 1 nM concentration was carried out as described above. Nuclei were visualized by staining of DNA with SiR-DNA reagent (Cytoskeleton) (0.25 μM) for 6 h. Cells were cultured in CellView 3.5 cm glass-bottomed dishes (Greiner). Time-lapse images were obtained using a Leica TCS SP5 DMI6000 CS Confocal Microscope between 48 and 72 h post transfection.

Transfection of siRNAs (siADAR1-1) into HeLa cells at 1 nM concentration was carried out as described above. After incubation for 24 h, the culture medium was replaced with a fresh medium containing CellLight Tubulin-GFP and BacMam 2.0 (Thermo Fisher Scientific). Nuclei were stained with SiR-DNA reagent (Cytoskeleton) at 0.25 μM for 6 h. Cells were cultured on Ibidi μDish 3.5 cm. After 72 h, cells were fixed with 4% paraformaldehyde and soaked in Dulbecco’s PBS. Microscopic images were obtained by using a Leica TCS SP5 DMI6000 CS Confocal Microscope and LAS X software (Leica), equipped with ultraviolet 405 diode, Argon, DPS3561, and HeNe594 lasers. Fluorescent images were captured with a 40× lens with a 512 × 512 frame. For multicolor experiments, the following wavelength settings were used: Tubulin-GFP (Ex 488 nm/Em 498–630 nm) and SiR-DNA reagent (Ex 647 nm/Em 657–800 nm). Nuclear morphological analysis was performed using 4′,6-diamidino-2-phenylindole (DAPI)-stained HeLa cells.