U-46619 br Materials and methods br
Materials and methods
Caudatin (Purity: 98%) was purchased from Shenzhen Medherb Biotechnology Co., Ltd. (Shenzhen, China). The JNK Inhibitor SP600125 was purchased from Merck Chemicals (Darmstadt, Germany). The p38 MAPK inhibitor SB203580 was obtained from Santa Cruz Biotechnology (Shanghai, China). Triptolide (Purity: 98%) was purchased from Selleck (Shanghai, China).
Antibodies specific to β-actin and p-Cdc2 were obtained from Sigma-Aldrich. Anti-Caspase-8, Caspase-9, PARP, DR5, p-p38 CHOP, PERK and BIP U-46619 were purchased from Cell Signaling Technology (Shanghai, China). Mouse anti-p27 and p-JNK were pur-chased from BD Biosciences (San Jose, CA, USA). Anti-p21, p53, cyclinB1, Cdc2, Cdk4, ERK, p-ERK, p38, JNK, ATF4 and DR4 antibodies were purchased from Santa Cruz Biotechnology.
The human breast cancer cell lines MDA-MB-231 and MCF-7 were purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). MDA-MB-231 cells were cultured at 37 °C under 5% CO2 in L-15 medium (Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS, GIBCO, USA). MCF-7 cells were routinely cultured in Dulbecco's modified Eagle's medium (DMEM, Invitrogen) supplemented with 10% FBS. r> Cytotoxicity assay
The cytotoxicity of caudatin was measured by the cell counting kit-8 (CCK-8) (Dojindo Laboratories, Japan) assay according to the manu-facturer's protocols. Briefly, cells were plated in 96-well plates in a total volume of 100 μl of medium per well. After 24 h of treatment, 10 µl of CCK-8 was added to each well, and the cells were then cultured at 37 °C for 1 h. Absorbance was measured at a wavelength of 450 nm by using a microplate reader. Assays were repeated three times.
Fig. 1. Caudatin inhibits the viability of human breast cancer cells. MDA-MB-231 and MCF-7 cells were incubated with diﬀerent concentrations of caudatin for 24 h and cell viability was measured using a CCK-8 assay. *p < 0.05, **p < 0.01 compared with the control.
Cell cycle analysis
Cells were harvested with trypsin, washed with phosphate-buﬀered saline (PBS) and fixed with 75% ethanol for 12 h. After the removal of ethanol by centrifugation, the fixed cells were incubated with 50 μg/ml RNase A and stained with 20 μg/ml PI at 37 °C for 30 min. The cell cycle distribution under each treatment was determined by flow cytometry analysis, and the data were analyzed by using CellQuest analysis soft-ware (Becton Dickinson, USA).
Cell apoptosis detection
Apoptotic cells were detected by TUNEL staining (Roche, IN, USA) according to the manufacturer's instructions. Briefly, cells were fixed with 4% paraformaldehyde, followed by permeabilization with 0.1% Triton X-100 before incubation in the TUNEL reaction mixture. Then, the cells were incubated with the TUNEL reaction mixture at 37 °C for 1 h and stained with DAPI. The stained cells (green) were visualized under a fluorescence microscope.
CHOP small interfering RNA (siRNA), DR5 siRNA and negative control siRNA were obtained from Santa Cruz Biotechnology. Cells were transfected with siRNA at a final concentration of 10 nM using the Lipofectamine™ RNAi MAX Reagent (Invitrogen) in accordance with the manufacturer's instructions.
Western blotting analysis
Cells were washed with cold PBS and lysed on ice with RIPA buﬀer supplemented with proteases and phosphatases inhibitors. The lysates were subsequently centrifuged at 4 °C for 20 min, and the supernatant fractions were collected. Equal amounts of proteins were subjected to SDS-PAGE and then transferred to polyvinylidene fluoride membranes. The membranes were blocked with 5% milk for 2 h and then incubated with appropriate primary antibodies overnight at 4 °C. Following 3 washes in TBST, the membranes were incubated with (HRP)-conjugated secondary antibodies for 1 h at room temperature. Enhanced chemilu-minescence with ECL detection reagents (Millipore) was utilized to detect the resultant signals.
All experiments were performed in triplicate. Statistical analysis was performed using SPSS 24.0 software. The results are shown as the mean ± standard deviation (SD), and p values < 0.05 were considered statistically significant.
Fig. 2. Caudatin induces cell cycle arrest in human breast cancer cells. (A) Cells were seeded in six-well plate and incubated with caudatin for 24 h. Cells cycle distribution was determined by flow cytometry. *p < 0.05, **p < 0.01 compared with the control. (B) Cells were treated with increasing doses of caudatin, and western blotting analysis was used to detect the cell cycle-related protein expression. β-actin was used as a loading control.