Fv10 asw ver 2.1

Cells were fixed with 4% paraformaldehyde in PBS at 1-h intervals, permeabilized with 0.5% Triton X-100, and blocked with 3% BSA for 30 min. Incubation with primary antibodies against PTEN (Bioworld Technology, Inc, MN, USA) was done overnight at 4°C. Then, the nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich) 20 min before imaging. A laser scanning confocal microscope FV10-ASW (Ver 2.1) (Olympus Corp, MPE FV1000, Tokyo, Japan) was used for colocalization analysis.

Cells were rinsed with PBS and fixed with 4% paraformaldehyde in phosphate buffer (pH 7.4) for 20 min at room temperature. After washing with PBST, the cells were blocked with 3% bovine serum albumin in PBST for 1 h, incubated with anti-p53 antibody/anti-Recql4 antibody (Santa Cruz Biotechnology) overnight at 4°C, and then washed with PBST. Tetramethylrhodamine-labeled anti-mouse/anti-rabbit IgG antibody (Rockland, Limerick, PA, USA) was then added and the cells incubated for 1 h. Mitochondria were identified by Mito Red (KeyGen) according to the manufacturer's instructions. Finally, the cells were rinsed with PBST and exposed to DAPI for 15 min to stain the nuclei. After washing with PBS, the cells were examined under a laser scanning confocal microscope FV10-ASW [Ver 2.1] (Olympus Corp, MPE FV1000) for co-localization analysis.