P cdk1

Cells were seeded in 6 well plates at 250 × 103 cells/well density and were treated with Rig. Cells were seeded and treated as described in the trypan blue vital count paragraph. After incubation with the drugs, cells were lysed with a lysis buffer (5 mM Hepes pH 7.5, 150 mM NaCl, 10% Glycerol, 1% Triton X100, 1.5 mM MgCl2, 5 mM EGTA, 0.1 M PMSF, 1% aprotinine, 0.1 M Na-pyrophosphate, 0.5 M Na3VO4). Protein content was quantified using the Bradford method. 10 µg of proteins were separated in SDS-PAGE gel and transferred to a nitrocellulose membrane. Western blotting against p53 (1:1000, #9282, Cell Signaling, Danvers, MA, USA), EMI1 (1:1000, #sc-365212, Santa Cruz Biotechnology, Dallas, TX, USA), Cyclin B (1:1000, #sc-166210, Santa Cruz Biotechnology, Dallas, TX, USA), P-CDK1 (1:1000, #9111, Cell Signaling, Danvers, MA, USA), CDK1 (1:1000, #MA5-15629, Invitrogen, Waltham, MA, USA), and PLK1 (1:1000, #sc-17783, Santa Cruz Biotechnology, Dallas, TX, USA) and p21 (1:1000, #2947, Cell Signaling, Danvers, MA, USA) was performed following the manufacturer’s instructions. Immunoreactive proteins were visualized using an ECL chemiluminescence system (Amersham, Marlborough, MA, USA) and bands were quantified using ImageJ 1.52p software.

Protein extract, western blot and co-IP were performed as described [36 (link)]. Antibodies used were as followings: mouse monoclonal anti-TAZ (1:1000) antibody from BD Biosciences; mouse monoclonal anti-β-actin (1:10, 000) and anti-FLAG (M2; 1:500) antibodies from Sigma; rabbit polyclonal anti-HA (1:1000) and anti-Thiophosphate ester (1:1000) antibodies from Abcam; rabbit polyclonal anti-MST1, MST2, LATS1, CyclinB1, Cdk1, cleaved-PARP (c-PARP), BCL-2, and pCdk1 (all diluted at 1:1000) antibodies from Cell Signalling Technology.

Cells were lysed and total protein extracted in RIPA lysis buffer (Applygen, Beijing, China) supplemented with protease inhibitors (Roche, Indianapolis, IN) and phosphatase inhibitors (Applygen, Beijing, China) at 4 °C for 30 min. The cell lysate was centrifuged at 12,500 rpm at 129,50 g-force for 15 min at 4 °C and protein concentrations were measured by BCA method (Beyotime, Guangzhou, China). Proteins were separated by 10% SDS-PAGE and transferred to a PVDF membrane (Millipore, Billerica, MA). After blocking with 5% fat-free milk in TBST for 1 h, membranes were immunoblotted with primary antibodies to caspase3, cleaved-caspase3, PARP, cleaved-PARP, p21, p-RB, PLK1, p-CDK1, and Aurora B (Cell Signaling Technology); p53, FOXM1, Aurora A, CDC-25C, CDK-1, cyclin B1, TOP2A, and GAPDH (Proteintech) at the appropriate dilutions with gentle shaking overnight at 4 °C. Blots were incubated with goat anti-mouse or anti-rabbit HRP-conjugated secondary antibody (1:3000; CWBIO, Beijing, China) and bands were visualized using an enhanced chemiluminescence, eECL Western Blot Kit (CWBIO, Beijing, China).

The following antibodies were used: Pol II CTD S2 (Bethyl cat# A300-654A, 1:10,000); Pol II CTD S5 (Bethyl cat# A300-655A, 1:10,000); Pol II (Santa Cruz cat# sc-899, 1:1000); cleaved PARP (Cell Signaling cat# 9541, 1:1000); Cleaved Caspase-3 (Cell Signaling cat# 9661, 1:1000); GAPDH (Cell Signaling cat# 2118S, 1:4000); CDK12 (Cell Signaling cat# 11973S, 1:1000); CDK7 (Santa Cruz cat# sc-365075, 1: 1000); MYCN (Cell Signaling cat# 9405S, 1: 1000); CDK1 (Santa Cruz cat# sc-53219, 1:1000), pCDK1 (Cell Signaling cat# 9114, 1:1000); CDK2 (Cell Signaling cat#2546, 1:1000), pCDK2 (Cell Signaling cat# 2561, 1:1000); cyclin H (Santa Cruz #sc-609, m1:1000).

Western blot analysis was performed as described previously^{30 (link)}. Briefly, cells were treated with CWPHs (P-4.3, 231 μg/ml), incubated over a period of 24 h, 48 h and 72 h. The primary antibodies used were p53, p21, CDK1, p-CDK1 (Tyr 15), Cyclin B1, p-histoneH3 and GAPDH (Cell Signalling Technology). Further blots were treated with horseradish peroxidase–conjugated antibody. Protein bands were detected using WesternSure Chemiluminescent Substrate (LI-COR) and captured by a Versadoc imager system (Biorad, USA). Quantification of the proteins was performed using the ImageJ software with GAPDH as the internal control.

β-Elemene was obtained from the National Institutes for Food and Drug Control (NIFDC, Beijing, China) Antibodies against β-actin, GAPDH, CD133, CD44, Prx-1, γ-H2AX, RAD51, CDC6 and ki67 were purchased from Proteintech. Antibodies for iNOS (ab129372) were obtained from Abcam while those for E-cadherin, Vimentin, β-catenin, N-cadherin, p-CDK1, p-Rb, P21, p-IkB α, IKK α/β, p-IKK α/β were purchased from Cell Signaling Technology.