Sp5 confocal microscopy system

NIH3T3 cells were typically seeded at 50% on sterile glass coverslips before 16–18 h. After transfected with GFP-fused TTP expression vector using Lipofectamine 2000 (Thermo) for 24 h and treated with 20 ng/ml of leptomycin B (LMB) for 6 h, cells were washed briefly with PBS and then fixed in 4% paraformaldehyde/PBS for 30 min at room temperature. After gently washed twice with PBS (5 min for each), cells were permeabilized with 0.1% Triton X-100/PBS for 10 min at room temperature. Following another two times washed with PBS (5 min for each), blocked the cells for 30 min with PBS containing 1% BSA. Then the cells were incubated with appropriately diluted DDX6 antibody overnight at 4 °C, washed with PBS three times (10 min for each), further incubated with secondary antibody (Alexa Fluor 594 goat anti-rabbit Ig, molecular probe) and DAPI (Sigma-Aldrich) for 1.5 h in the dark, and washed with PBS three times (10 min for each) in the dark. Mount in mounting fluid and store at − 20 °C until detected by Leica SP5 confocal Microscopy system.

Cell death was determined by TUNEL assay. Frozen sections and fixed cultured cells were stained with TnT antibodies and incubated with a TUNEL stain (In Situ Cell Death Detection Kit, POD, Roche Diagnostics GmbH, Mannheim, Germany). The sections were then co-stained with DAPI and visualised using a Leica SP5 confocal microscopy system. The images were analysed using ImageJ 2.1 software.

Cells were fixed with 4% paraformaldehyde in PBS (−) for 15 min at room temperature, then permeabilized and blocked with blocking buffer (1% BSA, 0.3% Triton X-100 in PBS(−)) for 1 h at room temperature. Anti-FOXO3a antibody (1:300) was incubated overnight at 4 °C and an anti-rabbit IgG antibody coupled with Alexa Fluor 488 was used for detection. DRAQ5 was used to counter-stain nuclei. Images were acquired using a Leica SP5 confocal microscopy system.