Pard3

Protease inhibitors (Boster, Wuhan, China) were added to cell lysates on ice for 20 min. Lysates were centrifuged at 14 000 rpm for 10 min at 4°C. Proteins (50 μg) were boiled for 5 min and separated by SDS-PAGE. Separated proteins were transferred to a polyvinylidene fluoride (PVDF) membrane and blocked for 1 h at room temperature. Blots were incubated with rabbit polyclonal antibody against PARD3 (Abcam, Cambridge, MA, USA) or β-actin (Sangon, Shanghai, China) at 4°C overnight, followed by incubation with the secondary antibody (ZSGB-BIO, Beijing, China) at room temperature for 1 h. A chemiluminescent substrate (Thermo Fisher Scientific, Waltham, MA, USA) was added. Quantity One software was used to quantify the intensity of each band. β-actin was used as control.

Twenty-four hours after treatment with a GMF according to the above mentioned method, cells were fixed with cold 4% formaldehyde for 10 minutes at room temperature and subsequently incubated with phosphate-buffered saline (PBS) containing 0.3% triton X-100 and 2% bovine serum albumin for 30 minutes at room temperature. This was followed by incubation with primary antibodies against Ki67 (Abcam, Cambridge, MA, USA), Cleaved Caspase-3 (Cell Signaling Technology, Danvers, MA, USA), and PARD3 (Abcam) overnight at 4°C. After rinsing with PBS, cells were incubated with Alexa594-conjugated goat anti-rabbit IgG antibody. After a further rinse with PBS, the cells were incubated with 1 μg/mL of 4′,6-diamidino-2-phenylindole (Sigma-Aldrich, St Louis, MO, USA) for nuclear staining.