Cyclin d1

The human TNBC cell lines MDA-MB-231 and MDA-MB-468 were from ATCC and stored in our laboratory. The docetaxel or vinorelbine-resistant MDA-MB-231 cell lines (MDA-MB-231/DR or MDA-MB-231/VR) were treated and established with docetaxel or vinorelbine for about 6 months in our laboratory. All these cell lines were cultured in DMEM medium supplemented with 10% fetal bovine serum (HyClone, USA) in 5% CO₂ at 37 °C. Docetaxel and vinorelbine were purchased from Aosaikang and Haosen in China, respectively. Aspirin was purchased from Sigma in USA. The following antibodies were used for IHC and western blotting: YAP (Abcam, UK), β-catenin (Santa Cruze, USA), β-TrCP, Bcl-l2, Bax (CST, USA), C-Myc, CyclinD1 (Epitomics, USA), Ki67 (MXB biotechnologies, China), GAPDH (Biostar, China).

Total proteins from the cells or tissues were extracted with RIPA lysis buffer. Protein concentrations were measured by use of the BCA reagent kit (Merck). The proteins were resolved by SDS-PAGE and transferred to a PVDF membrane, which was probed with specific primary antibodies against FBXL11 (Abcam), FBXO31 (Abcam), CyclinE1(Cell Signaling Technology), P21 (Epitomics), CyclinD1 (Epitomics),CyclinD2 (Santa cruz biotechnology),CyclinD3 (Santa cruz biotechnology) followed by anti-mouse or rabbit horseradish peroxidase-conjugated IgG (Bio-Rad) and developed with the chemiluminescence method (ECL, Millpore). β-actin (Sigma-Aldrich) servedas a loading control.

Oridonin was purchased from Sigma-Aldrich (St. Louis, MO, USA). For in vitro studies, Oridonin was dissolved in dimethyl sulfoxide (DMSO) to create a stock solution (0.1 mol/L), which was stored at −20°C. To prepare working solutions, the stock solution was further diluted with culture media to yield the desired Oridonin concentration. Control cells were treated with an equal volume of vehicle. The DMSO concentration was kept below 0.1% in cell culture and did not have any detectable effect on cell growth or cell death.
3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT), Hoechst 33342, annexin V-FITC, propidium iodide (PI), and Rhodamine 123 were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Primary antibodies against caspase-3, caspase-9, NF-κB, Bax, Bcl-2, PARP-1, cytochrome c, β-actin, and secondary antibodies (goat-anti-rabbit or goat-anti-mouse) were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies against cyclin A, cyclin B1, and cyclin D1 were purchased from Epitomics (Burlingame, CA, USA).

Western blot was performed as described previously [34 (link)]. A total of 40μg of protein lysates were separated by sodium dodecyl sulphate/polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto a polyvinylidene difluoride (PVDF) membrane (Bio-Rad, Hercules, CA, USA). The primary antibodies were against: DKK2 (ab38594, Abcam), active β-catenin (#05-665; Merck Millipore, Billerica, MD, USA), total β-catenin (#2677; Cell Signaling Technology, Danvers, MA, USA), c-Myc (#1472-1; Epitomics, Cambridge, MA, USA), cyclin D1 (#1677-1; Epitomics), occludin (ab31721; Abcam), vimentin (#2707-1; Epitomics), Ecad (#1702-1; Epitomics), N-cadherin (ab98952;Abcam), and β-actin (LK-ab008-100; Liankebio, China), and GAPDH (#AE082046; Beijing Biosynthesis Biotechnology, Beijing, China) was used as a control. Proteins were visualized using an enhanced chemiluminescence kit (Amersham Pharmacia Biotech, Piscataway, NJ, USA).

All paraffin-embedded specimens were cut into 4-μm sections and dried at 60 °C for 2 h, deparaffinised and dehydrated. The sections were then boiled in 0.01 M citric acid buffer solution (pH 6.0) for 1.5 min at high pressure. After being incubated with 3% hydrogen superoxide for 20 minutes to quench endogenous peroxidase activity and 10% normal goat serum to block non-specific binding, the sections were incubated overnight at 4 °C with polyclonal rabbit anti-human AGR2 (Cell Signaling Technology, 1:200), Survivin (Cell Signaling Technology, 1:200), Cyclin D1 (Epitomics, 1:200), ALDH1 (Proteintech gruop, 1:100), (Epitomics, 1:200), OCT4 (Proteintech gruop, 1:100), Slug (Cell Signaling Technology, 1:200). Then, the sections were incubated with a secondary biotinylated immunoglobulin G antibody solution and an avidin-biotin-peroxidase reagent. After being washed with phosphate buffer saline, the sections were incubated with 3,3′-diaminobenzidine tetrachloride and then lightly counterstained with Mayer's haematoxylin.

Cells were lysed in ice-cold whole cell lysis buffer(50 mM Tris–HCl, pH 8.0, 4 M urea and 1% Triton X-100) supplemented with complete protease inhibitor Cocktail (Roche Diagnostics, 04693132001). Whole cell lysates with equal protein were resolved by SDS-PAGE and transferred to nitrocellulose membrane. After blocking with 5% milk in Tris-buffered saline plus 0.02% Tween-20 (TBST), membranes were incubated with the following antibodies: cyclin D1 (Epitomics, 1677–1), cyclin D3 (Epitomics, 1846–1), E2F1 (Cell Signaling, 3742), cyclin E (Santa Cruz, sc-481), cyclin A (Santa Cruz, sc-596), CDK2 (Cell Signaling, 2546), CDK4 (Cell Signaling, 2906); phosphor-Rb (Ser 780) (Cell Signaling, 3590), phosphor-Rb (Ser-811) (ABclonal Biotechnology, AP0089), total Rb (Cell Signaling, 9309) and GAPDH (Proteintech, 10494-1-AP). Membranes were then incubated with horseradish peroxidase-coupled specific secondary anti-mouse (KPL, 074–1806) or anti-rabbit antibodies (KPL, 474–1506). Protein bands were visualized using ECL blotting detection reagents (KPL, 54-61-00).