br Apoptosis assay br T
2.5. Apoptosis assay
4T1 cells were cultured in the presence or absence of ART (100 μM) for 48 h in 96-well plates at a concentration of 1 × 106 cells/mL. To detect apoptosis, the cells were stained using an Annexin-V Apoptosis Detection kit I (BD Biosciences) and subsequently evaluated by flow cytometry. The results were analyzed using FlowJo v7.6.2 and GraphPad Prism 6.0.1 software.
Spleens and tumors were excised from the 4T1 tumor-bearing mice. Single cell suspensions were produced by homogenizing the spleen samples, and the tumors were minced and subsequently digested with 500 U/mL collagenase type IV (Sigma) for 1 h at 37 °C with agitation. The resulting single cell suspensions were suspended in RPMI 1640 with 10% fetal calf serum (FCS, Gibco, England).
To stain myeloid-derived suppressor cells (MDSCs), anti-CD11b FITC (clone M1/70, BD Biosciences) and anti-Gr-1 APC (clone RB6-8C5, BD Biosciences) were added to cells suspended in PBS with 3% FCS and incubated for 30 min. Anti-CD4 FITC (clone H129.19, BD Biosciences) and anti-CD25 PE (clone PC61, BD Biosciences) Calpain Inhibitor I were used to stain the cell surface of regulatory T cells (Tregs). The cells were then fixed and permeabilized for intracellular staining, and an anti-Foxp3 APC (clone FJK16s, eBioscience) antibody was added. To analyze CD4+ IFN-γ+ T cells and cytotoxic T lymphocytes (CTLs) expressing gran-zyme B, anti-CD4 FITC (clone H129.19, BD Biosciences), anti-CD8a PerCP (clone 53–6.7, BD Biosciences), and anti-IFN-γ APC (clone XMG1.2, BD Biosciences) were added to the cells and incubated for 30 min. Cells were then fixed and permeabilized, and intracellular staining was performed using an anti-granzyme B PE antibody (clone NGZB, eBioscience). All staining reactions were performed in a final volume of 100 μl at 4 °C. Data were acquired using a FACS Calibur flow cytometer (BD Biosciences, San Diego, CA, USA) and analyzed using FlowJo v7.6.2 software (Tree Star Inc., Ashland, OR, USA).
A commercial ELISA kit (R&D Systems, Minneapolis, MN, USA) was used to analyze TGF-β levels in the cell culture supernatants. All steps were performed according to the manufacturer's instructions, and OD values were measured at 450 nm using a microplate reader. A standard curve was generated using known concentrations of recombinant cy-tokines, and this was used to calculate the TGF-β concentrations of the
Fig. 1. ART treatment inhibited the proliferation of 4T1 cells in vitro. 4 T1 cells were treated with various concentrations of ART for 24, 48, and 72 h. Cell pro-liferation was then was determined by MTT assay. TGF-β levels in the supernatant were detected by ELISA after treatment with ART for 48 h. Apoptosis was measured by flow cytometry. Results are shown as mean ± SD. P < 0.01 and P < 0.001 compared to the control group.
Fig. 2. ART inhibited the growth of 4T1 tumors and prolonged the survival of 4T1 TB mice. Tumors were removed from mice after ART treatment and body weights were compared (A). Tumor volume (B) and body weight (C) curves at the end of treatment. Survival curves (D) of 4T1 TB mice (n = 7). The results are shown as mean ± SD. P < 0.05 and P < 0.001 compared to the control group.
Fig. 3. ART treatment decreased the frequencies MDSCs and Tregs in the spleens and tumors of 4T1 TB mice. Flow cytometric analysis of MDSCs (CD11b+ Gr-1+) and Tregs (CD4+ CD25+ Foxp3+) in both the spleen and tumor (A and B). P < 0.05 compared to the control group.
2.8. RNA isolation and real-time RT-PCR
Trizol reagent (Invitrogen, Carlsbad, CA) was used to extract total RNA from excised tumors (~100 mg) per the manufacturer's instruc-tions. The RNA was then quantified at 260 nm using a UV–VIS spec-trophotometer (PYE-UNICAM, USA). To remove any contaminant genomic DNA, the RNA was treated with DNaseI and cDNA was syn-thesized using the PrimeScript™ RT reagent kit with gDNA Eraser kit (Takara, China). PCR was then performed using the cDNA as a tem-plate. Table 1 shows the specific oligonucleotide primers used for the PCR reaction. Quantitative PCR was performed on an ABI 7500 real-time PCR system (ABI, USA) using a SYBR® Premix Ex Taq™ reagent kit (Takara) according to the manufacturers' instructions. β-actin amplifi-cation served as an internal control. Gene expression was quantified using the 2− CT method.
2.9. Statistical analysis
Results are presented as the mean ± standard deviation of three experiments. One-way ANOVA and two-tailed Student's t-tests were used to test for statistical significance. The Kaplan–Meier method was
used to calculate survival analysis. P < 0.05 was considered statisti-cally significant.