Cell lines and cell culture Mouse macrophage cell line RAW 264.7, the human monocyte cell line THP-1, human colon cell line HCT116 and mouse colon cell line CT26 were kindly provided by the Cell Bank of Type Culture Collection of Chinese Academy of Sciences (Shanghai, China). RAW264.7 Cell were maintained in DMEM, while THP-1, HCT116 and CT26 were maintained in RPMI-1640. All cultures were supplemented with 10% fetal bovine serum, 100 IU/ml penicillin and 100 µg/ml streptomycin. The macrophages were polarized into M2 by 20 ng /mL IL-4/IL-13 for 24 h. Phorbol-12-Myristate-13-acretate (PMA) was taken to utilized to differentiate THP-1 cells.
CCK-8 assays CT-26 and HCT116 were seed in a 96-well plate with a density of 5 × 103. Cell Viability Assay was determined using CCK8 in accordance with the manufacture’ s instruction. The absorbance was measured at 450 nm with a microplate reader.
Flow cytometry RAW264.7 and THP-1 cells were stained with the following fluorochrome-conjugated antibodies: anti-CD206-PE (Biolegend, USA), anti-CD86-FITC (Biolegend, USA). Single-cell suspension from tumors were performed using mouse tumor dissociation kit (Miltenyi Biotec, Germany). Stained cells were analyzed by FACS. Data analysis was performed using FlowJo software.
Colony formation CT-26 and HCT116 were seed in a 12-well plate with a density of 1×103. The fresh medium was changed every day to keep the cell growing. After 10 days, the colonies were fixed with 4% paraformaldehyde and dyed with crystal violet (Beyotime, China).
RT-PCR analysis RNA was firstly isolated from cell using MolPure® cell RNA Kit (Yesen, China). mRNA expression was performed using SYBR Green PC Master Mix (Yesen, China). Reaction was carried out using RT-PCR kits (Applied Biosystems, Canada). Expression of genes were analyzed as RQ = 2 − ΔΔCt. Primer sequences used for RT-PCR are listed in Table 1.
Microscale Thermophoresis The interaction between Cucurbitacin B and STAT3 was detected by Microscale Thermophoresis using the Nano Temper Monolith NT.115 instrument. Each measurement consists of 16 traction mixtures where fluorescent-labeled stat3, and two-fold diluted cucurbitacin B ranging from 100µM to 3.1nM was used. The MO. affinity Analysis v2.3 software was used to measure the Kd.
Wound healing assay CT-26 and HCT116 cells were planted in a 12-well plate (5 × 106) and grown until 80% confluent,and a wound was made by dragging a plastic pipette tip across the cell surface. Then wound healing image was photographed again the next day using microscope. The area of the wound was measured with image J software.
Migration assay The migration assay was conducted in a 24-well cell culture chamber employing inserts with 8-µm pores (Corning, NY, USA). Inserts that contain 2 × 105 CT-26 and HCT116 cells were transferred to wells containing 5 × 105 M0 macrophages, M2 macrophages cultured with or without cucurbitacin B for 24 h. A cotton swab cell was removed on the top well. After fixation in 4% paraformaldehyde, the filters were stained with crystal violet for 15 min.
Western blot analysis CT-26, HCT116, RAW264.7 and THP-1 cells were lysed in RIPA buffer (Thermo, MA, USA). The cell lysates were separated on 10% or 12.5% SDS-PAGE gels. The antibodies used were against JAK2, p-JAK2, STAT3, p-STAT3. (Cell Signaling Technology, USA).
Immunofluorescence staining Cells that treat with above way were fixed with 4% paraformaldehyde (Beyotime, China) for 30 min and were incubated with STAT3 antibody. The image was pictured under the confocal microscope.
Cell apoptosis assay Cell apoptosis was evaluated using an Annexin V- FITC Kit (Biolegend, USA). Cells were analyzed using a CytExpert flow cytometer (Beckman Coulter, USA).
C57BL/6 and BALB/c murine colon cancer modelIn vivo experiment was conducted in compliance with the relevant laws and institutional guidelines. In C57BL/6 model: CT-26 (8 × 105) in 0.2 mL were injected subcutaneously into the flank of each mouse. After 1 d, mice were intraperitoneally injected administrated with 0.2 mL of cucurbitacin B (0.5 mg/kg) and 0.2 mL of Cucurbitacin B 1 mg/kg) for 25 d according to previous study[20]. Control group received equal volumes of normal saline (NS). On day 14 the animals were euthanized.
Table 1
Mouse Ym-1
|
Forward
|
5’-AGAAGGGAGTTTCAAACCTGGT-3’
|
Reverse
|
5’-CTCTTGCTGATGTGTGTAAGTGA-3’
|
Mouse Fizz1
|
Forward
|
5’-AGTCCCTGCCCTTTGTACACA-3’
|
Reverse
|
5’-CGATCCGAGGGCCTCACTA-3’
|
Mouse Arg-1
|
Forward
|
5’-CTCCAAGCCAAAGTCCTTAGAG-3’
|
Reverse
|
5’-GGAGCTGTCATTAGGGACATCA-3’
|
Mouse IL-10
|
Forward
|
5’-CTTACTGACTGGCATGAGGATCA-3’
|
Reverse
|
5’-GCAGCTCTAGGAGCATGTGG-3’
|
Human Ym-1
|
Forward
|
5’-TGCCACCTCCAGTCCAGTGA-3’
|
Reverse
|
5’-ATGCCGTAGAGCGTCACATC-3’
|
Human Fizz1
|
Forward
|
5’-CGTCCTCTTGCCTCCTTCTC-3’
|
Reverse
|
5’-ACAAGCACAGCCAGTGACAG-3’
|
Human Arg-1
|
Forward
|
5’-TGCCACCTCCAGTCCAGTGA-3’
|
Reverse
|
5’-GCATCCAGCTTGACCAGAGA-3’
|
Human IL-10
|
Forward
|
5’-GATCTCCGAGATGCCTTCAG-3’
|
Reverse
|
5’-ATCGATGACAGCGCCGTAGC-3’
|
CT-26 cells/effluc cells (1×106) intravenously were injected into BALB/c the for a tumor progression study. Luminescent tumor images were monitored after in the intraperitoneal injection of Luciferin for 10min and detected by a Xenogeny IVIS Lumina II imaging system.
Immunofluorescent staining Tumor tissues were made into slices. Then, all tumor slices were incubated with anti-Ki67 antibody, anti-Caspase-3 antibody and anti-CD206 antibody (1:200) (Cell Signaling Technology, USA), followed by incubation with secondary antibodies. Images were taken through a light microscope.
Statistical analysis
Values are expressed as the mean ±SD. Data were analyzed with GraphPad Prism software 8.0. Statistical analysis was performed by Unpaired Student’s t-test (two-tailed) and one-way ANOVA test. P<0.05(*) was considered statistically significant.