Hoechst 33285

The cell culture medium (RPMI 1640), fetal bovine serum (FBS) and penicillin–streptomycin were purchased from Gibco BRL (life technolologies, Paisley, Scotland). The culture plates were obtained from Nunc (Denmark). Hoechst 33285 was purchased from Sigma Chemical Company (Germany). MTT assay kit was purchased from Roche (Germany). All antibodies, except β actin, including anti-Bcl2 and anti-Bax were purchased from Sigma (St Louis, MO, USA). β actin antibody was purchased from Alexis Biochemicals (San Diego, CA). Annexin-V-FITC kit was purchased from IQ product (Groningen, The Netherlands). All cell lines were obtained from Pasteur Institute of Iran (Tehran).

The nuclear relocation assay was carried out in cells seeded onto sterile glass coverslips. Cells co-expressing UAF1-mRFP with the different GFP- or YFP-tagged proteins were fixed with 3.7% formaldehyde in phosphate-buffered saline (PBS) for 30 min, incubated with Hoechst 33285 (Sigma) to visualize the nuclei, washed with PBS, and mounted onto microscope slides using Vectashield (Vector laboratories). Single-slice images were acquired using an Olympus Fluoview FV500 confocal microscope. Sequential acquisition of each fluorochrome was performed in order to avoid overlapping of fluorescent emission spectra. For for live cell imaging, cells were grown in 35 mm ibiTreat μ-dish slides (Ibidi), transfected with the indicated plasmids and examined using a Zeiss ApoTome.2 microscope. Semiquantitative analysis of nuclear relocation assay samples was carried out by determining the nucleocytoplasmic localization of UAF1-mRFP in at least 100 co-transfected cells per slide using a Zeiss Axioskop fluorescence microscope. Slides were coded to ensure unbiased scoring, and examined by two independent observers.