Protein phosphatase inhibitor

OSW-1 (C47H68O15, purity ≥95%) was purchased from Cayman Chemical Company (Ann Arbor, Michigan, United States), dissolved in dimethyl-sulfoxide DMSO (Sigma-Aldrich, St. Louis, MO, United States) then stored in the dark at − 20°C. OSW-1 was diluted with cell culture medium to the required concentrations, with DMSO concentration <0.1%, to avoid side effects. Dulbecco’s modified Eagle medium (DMEM) and fetal bovine serum (FBS) were purchased from Thermo Fisher Scientific (Gibco, United States). Cell counting Kit-8 (CCK-8), RNase, penicillin/streptomycin, propidium iodide, Annexin V-FITC Apoptosis Detection Kit, RIPA lysate, protease inhibitor, protein phosphatase inhibitor, BCA protein assay kit, and electrochemiluminescence (ECL) kit were purchased from Beyotime Biotechnology Company (Shanghai, China). 740Y-P was purchased from TargetMol (Shanghai, China). Primary antibodies against P21 (ab109520), cyclin B1 (ab32053), CDK1 (ab133327), PARP-1 (ab191217), cleaved-PARP-1 (ab32064), cleaved caspase-3 (ab214430), beta Actin (ab8226), PI3K(ab191606), p-PI3K (ab278545), AKT1 (ab183556), and p-AKT1 (ab81283) were purchased from Abcam (Cambridge, MA, United Kingdom). Primary antibodies against cleaved caspase-9 (#9502) and secondary antibodies (goat anti-mouse IgG and goat anti-rabbit IgG, #98164 and #91196) were purchased from Cell Signaling Technology (Beverly, MA, United States).

The throat tissues were mechanically grinded in a glass homogenizer, the RIPA lysis buffer (Beyotime, Jiangsu, China) and protein phosphatase inhibitor (Beyotime, Jiangsu, China) were added to lysed the samples for 1 h on ice. In order to acquire supernatant, homogenates were centrifuged for 10 min at 12,000 rpm and 4 °C [29 (link)]. BCA protein test kit (Beyotime, Jiangsu, China) was used for the protein quantification. Then boiling the soluble extracts with Loading buffer (Beyotime, Jiangsu, China) for 10 min. The equal amount of protein exact of samples were electrophoresed on a 10% SDS-PAGE, then the electrophoretic proteins are transferred onto a PVDF. After blocking, membrane was probed with goat polyclonal antibodies against NF-κB p65 (1:2000; Proteintech, USA), COX-2 (1:1000; Proteintech, USA), GAPDH (1:5000; Proteintech, USA). The second day, rinsing the membrane with tris-buffered saline Tween (TBST) 3 times for 10 min, and incubating with goat anti-rabbit IgG (1:5000; Proteintech, USA), which can conjugate with appropriate secondary antibody HRP. Afterwards, cleaning the membrane again with TBST and treated with a chemiluminescence analysis kits (Beyotime, Jiangsu, China) for visualization. The bands were analyzed (relative to GAPDH expression) by using Gel-pro analyzer [30 (link)].

Total protein was extracted in RIPA lysis buffer (Beyotime Biotechnology, Shanghai, China) supplemented with phenylmethylsulfonyl fluoride (PMSF, Beyotime) and protein phosphatase inhibitor (Beyotime). The protein concentrations were measured using the BCA Protein Assay (Beyotime). The samples were separated on a 10% SDS-polyacrylamide gel and then transferred to a nitrocellulose membrane, which was blocked in 5% skim milk for 1 h and then incubated with primary antibodies at 4 °C overnight. After the washing steps, the membranes were incubated with HRP-labeled secondary antibodies for 1 h at 37 °C. The bands were visualized using Immobilon Western HRP (Millipore, Germany). Relative band densities of proteins in western blottings were normalized against GAPDH.