Primary antibodies for anti ki67

SiHa cells were exposed to DMSO, 3 or 10 µM TET at 37°C for 48 hours. Then, the incubated cells were washed three times with PBS, prefixed in 4% paraformaldehyde for 10 minutes at room temperature, and then fixed in pre-cold methanol for 10 minutes at −20°C. Later on, cells were incubated with primary antibodies for anti-Ki67 (Abcam, Cambridge, UK) (1:1,000), DAPI (1:1,000) at 4°C overnight. Subsequently, cells were incubated with secondary antibodies (Abcam) (1:2,000) at 37°C for 1 hour. The samples were observed by fluorescence microscope at once (Olympus, Tokyo, Japan).

SH-SY5Y cells were seeded into 24-well plates at a density of 4×10⁵ cells/well overnight. For treatment 1 (Figure 2A), SH-SY5Y cells were incubated with Andro (0 or 200 μM) for 12 hours. Then, the culture medium was changed and cells incubated with Bup (0 or 400 μM) for another 48 hours at 37°C. After that, cells were prefixed in 4% paraformaldehyde at room temperature for 20 minutes and fixed in cold methanol for 10 minutes at −20°C. Later, cells were incubated with primary antibodies for anti-Ki67 (1:1,000;
Abcam, Cambridge, UK) () and DAPI (1:1,000;
Abcam) at 4°C overnight.
For treatment 2 (Figure 5A), SH-SY5Y cells were incubated with Andro (0 or 200 μM) for 12 hours. Then, the culture medium was changed and cells incubated with 10 nM AZD5363 for 1 hour. Later, the culture medium was changed and cells incubated with Bup (0 or 400 μM) for another 48 hours. Next, cells were incubated with primary antibodies for anti-p-Akt (1:1,000;
Abcam) and DAPI (1:1,000) at 4°C overnight. After that, cells were incubated with goat antirabbit IgG secondary antibodies (1:5,000;
Abcam) at 37°C for 1 hour. Cells were visualized with fluorescence microscopy (Olympus, Tokyo, Japan).