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).
The human breast cancer nER negative SKBR-3 cells obtained from the National Infrastructure of Cell Line Resource (Beijing, China) were maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum, 100 μg/ml streptomycin and 100 U/ml penicillin at 37 °C in a 5% CO2 atmosphere.
MTT cell viability assay
MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) cell viability assay was performed as already described . Briefly, cells were seeded in 96-well plates at a density of 5 × 103 cells per well in the growth medium. After 12 h, the cells were treated with 1–10 μM CPT or 0.2% DMSO as a control. 24 h or 48 h later, 15 μL MTT reagent (1 mg/mL) was added into each well and the plates were incubated for an additional 4 h in the dark. Subsequently, the supernatant was removed and the cells were lysed in 150 μl DMSO. The optical density was at a measuring wavelength of 490 nm and a reference wavelength of 570 nm using a plate reader (Multiskan GO, ThermoFisher Scientific, USA).
Analysis of cell cycle distribution
After treatment with 5–10 μM CPT for 48 h, SKBR-3 cells were removed using 0.05% trypsin. Samples were then washed with phosphate-buffered saline (PBS) for 10 min and stained with 50 μg/ml PI (Sigma, USA) and 250 μg/ml RNase in PBS for 30 min at room temperature in the dark. Subsequently, fluorescence activated cell sorting analysis was performed by FCM (BD FACSCanto II, Becton, Dickinson and Company, NJ, USA).
Small interfering RNA (siRNA) transfection
SKBR-3 cells were seeded in 6-well cell culture plates. At 80–90% confluent, the cells were transfected with 33 nM GPER siRNA or labeled non-target siRNA as control using Lipofectamine® 2000 transfection reagent according to the manufacture’s instruction. After 24 h, the medium was replaced with fresh medium and the cells were cultured for an additional 24 h before Western blot analysis of GPER expression and MTT cell viability assay.
Western blot analysis
Exponentially growing cells were seeded in a 6-well plate. 24 h after seeding, the SKBR-3 cells in the logarithmic growth phase were incubated in the absence or presence of CPT for 48 h. 1 μM G-1 or G-15 was added together with CPT in G-1 or G-15 treating group. Then the cells were washed twice with ice-cold phosphate-buffered saline (PBS) and placed in RIPA buffer (Applygen, Beijing, China) supplemented with 1% protein phosphatase inhibitor (All-in-one,100×, Solarbio, Beijing, China) immediately prior to use. Total protein extracts were severally collected in different centrifuge tubes and centrifuged at 12000 rpm for 10 min at 4 °C. Supernatant was recovered subsequently. Then the protein concentration was quantified using bicinchoninic acid (BCA) protein assay kit (Solarbio, Beijing, China). Equal quantities of protein (50 μg) from each sample were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were blocked with 5% skim milk dissolved in tris-buffered saline with Tween-20 (TBS-T) (1X TBS, 0.1%Tween 20) for 2 h at room temperature. Western blot analyses were performed using the following primary antibody dilutions: Rabbit anti human cyclin A (1:1000), cyclin B (1:10,000), cyclin D (1:10,000), CDK2 (1:10,000), PI3K (1:1000), GPER (1:250) and mouse anti β-actin (1,10,000). All the above antibodies were diluted in 5% skim milk, respectively. In addition, rabbit anti human p-AKT was diluted for 1:2000 in 5% BSA according to introduction. The membranes were incubated with the above primary antibody dilutions overnight at 4 °C and then washed with TBST for 30 min and incubated with the secondary antibody, horseradish peroxidase-labeled goat anti-rabbit IgG or goat anti-mouse IgG (15,000, Proteintech Group, Inc., China) at 37 °C for 1 h. After additional washes, the membranes were incubated using an enhanced chemiluminescence kit (ECL, Applygen, Beijing, China) and scanned in the multifunctional molecular imaging system (Azure C-Series C600, USA). At last, the bands were collected and analyzed by Image J software (version 1.48, National Institutes of Health, USA).
Immunofluorescence (IF) assay
Cells were seeded in 8 well chamber slide system (Thermos Fisher, NY, USA) at a density of 1 × 104 per well. Then the cells in the logarithmic growth phase were treated with indicated concentration of CPT with or without G-1 or G-15. Following incubation for another 24 h, the cells were fixed with 4% paraformaldehyde for 30 min, permeabilized with 0.02% Triton-X-100 for 20 min and blocked with 5% goat serum for 30 min. Then the cells were incubated with specific primary antibodies at 4 °C overnight followed by fluorescein isothiocyanate (FITC) labeled secondary antibody for 1 h away from light at room temperature. After washed with PBS, DAPI was used for nucleus staining. Images were visualized using an inverted fluorescence microscope (IX71, Olympus, Japan) and the mean fluorescence intensity was analyzed by Image J software.
Data are expressed as mean ± S.D. of three independent tests at least for each experiment. To compare means among multiple groups, one-way ANOVA followed by the LSD-t multiple comparison test was performed in this study. The statistical analyses were performed using SPSS 20.0 (SPSS, Inc., Chicago, IL, USA) for Windows. The significant difference was confirmed with P < 0.05.