Cyclinb

SAS cells were lysed, separated by 6–15% SDS-PAGE, transferred to a nitrocellulose membrane and then blocked with skimmed milk. GAPDH expression served as the protein loading control. The following antibodies were used for immunoblotting (IB): anti-GAPDH, cyclin B, cyclin D1, CDK2, CDK4, and CDK1 were obtained from Abcam (Cambridge, MA, USA); anti-LC3 and ATG5 were obtained from Abgent (San Diego, CA, USA); anti-p53 and p27 were obtained from GeneTex (Irvine, CA, USA); anti-p62/SQSTM1 was obtained from MBL (Nagoya, Japan); anti-PARP, cytochrome C, Bax, Beclin 1, and cyclin E were obtained from Cell Signaling Technology (Ipswich, MA, USA); anti-phospho-IkBα (S32/S36) was obtained from R&D Systems (Minneapolis, MN, USA); anti-active caspase 3 was obtained from BioVision (San Francisco, CA, USA); anti- Bcl-2 and cyclin A were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA); and anti-p21 was obtained from Thermo (Waltham, MA, USA). Z-VAD-FMK and 3-methyladenine (3-MA) was obtained from Sigma-Aldrich (St. Louis, MO, USA). After mixing with 4× sample dye, the samples were heated at 95 °C for 5 min, placed on ice for 5 min, and subjected to a Western blot analysis.

Cryptotanshinone (CPT, purity>98%, molecular weight 296.36) was obtained from National Institutes for Food and Drug Control (Beijing, China). 2.964 mg CPT was dissolved in 1 ml dimethyl sulfoxide (DMSO, Sigma, USA) to make a 10 mM stock solution which was then added to the medium at the indicated concentrations. The methyl thiazolyl tetrazolium (MTT) was obtained from Keygen (Nanjing, China). RPMI-1640 medium and 0.05% trypsin were from Gibco (USA) and Fetal bovine serum was from Corning Cell Gro (Australia). Lipofectamine® 2000 was from Invitrogen (Grand Island, NY, USA). GPER specific agonist G-1 and antagonist G-15 were from Cayman Chemical (Michigan, USA). The following antibodies were used: GPER, PI3K(p85), cyclin A, cyclin B, cyclin D, CDK2 (Abcam, USA), phospho-Akt (p-AKT, Ser473, Cell signaling, Boston, MA, USA). GPER siRNA, non-target siRNA and PI3K inhibitor LY294002 were obtained from Santa Cruz Biotechnology (Texas USA). Fluorescein-Conjugated Goat anti-Rabbit IgG(H + L) was from ZSGB-BIO (Beijing, China) and DAPI was from Solarbio (Beijing, China).

Total protein was extracted from the cells using the M-PER mammalian protein extraction reagent or from tissues using the T-PER tissue protein extraction reagent (Pierce, IL, USA). Equal amounts of protein (12 μg per lane) as estimated by a bicinchoninic acid protein assay kit (Pierce, IL, USA) were separated by 11% SDS-PAGE and then transferred onto nitrocellulose membranes. The blots were probed with rabbit monoclonal antibodies against UbcH10 (1:200), KIAA0101 (1:500), BubR1 (1:600), Mad2 (1:250), CDC25(1:400), CDK1 (1:300), CDK7 (1:500), CyclinB (1:350) and β-actin (1:1000) (Abcam, CA, USA), followed by incubation with secondary HRP-conjugated goat anti-rabbit antibody (Abcam, CA, USA). After a washing step, the bands were detected by chemiluminescence and imaged on X-ray films. β-actin was used as an endogenous reference for normalization.

Total protein was isolated from liver tissue by the RIPA (Radio Immunoprecipitation Assay) lysate (Beyotime, Guangzhou, China). Protein bands were separated by 10% SDS-PEGA (sodium dodecyl sulfate polyacrylamide gel electrophoresis) (Beyotime, Guangzhou, China) and transferred by the PVDF (polyvinylidene fluoride) membrane (Roche, Shanghai). Using 5% defatted milk (Beyotime, Guangzhou, China) blocked nonspecific binding, and the primary antibody was incubated at 4 °C overnight. Antibodies against GLI1 and GLI2 were purchased from GeneTex; Cyclin D and Cyclin B from Abcam; and Bub1b, Cdc20 and CDK2 from Poteintech.