Huaier Aqueous acquired from Qidong GAITISNLI(Jiangsu, China). Adriaamycin purchased from Shanghai Macklin Biochemical. 740Y-P purchased from MCE Company. The in vitro trial concentration was 10mg/kg. SC79 purchased from MCE Company.
Cell Culture
MDA-MB-231 (Procell CL-0150, Human) were kindly provided by Procell Life Science &Technology Co.,Ltd (Wuhan, China). Cell STR typing of DNA from MDA-MB-231cell lines showed that no human cell cross-contamination was found in the cell lines. The cell line was cultured in high-glucose DMEM (Gibco, Thermo Fisher Scientific, Suzhou , China) supplemented with 10% FBS(Biological Industries, Beit-Haemek, Israel),Penicillin-Streptomycin double antibody solution(100X) at 37°C, with 5% CO2 in a humidifified incubator. Cells in logarithmic growth were used for all experiments.
Cell Counting Kit-8
Enhanced Cell Counting Kit-8 (CCK-8) is applied to detect cell proliferation. Cells were infused a 96-well plate with a thickness for 1×104 cells/well. It was cured with adriamycin and Huaier Aqueous for 0h, 24h and 48h, and 10µL of CCK-8 reagent (Saint-Bio, Shanghai, China) was applied to each well. The cell analysis microplate detection system (BioTek, Vermont, US) was performed to measure the absorbance at 450 nm.
Wound Healing Assay
Vaccinate cells under an 6-well plate for a thickness about 1x106 cells/well and society for a 37°C, 5% CO2 cell hatchery. Serum-free medium was applied and the culture was resumed for 12h. Slide the tip of a sterile 200ul pipette tip vertically from the top of the 6-well plate to the bottom. Then add the drug treatments of each group, take pictures under an optical microscope at 0h and 48h after scribing, and record the width of the scratch.
Cell Invasion Assays
Cells starved for 12h without serum. Dilute 50mg/L BD Matrigel (1:8) with serum-free medium and cover it on the upper surface of the bottom film of the chamber, and air-dry the chamber toward 4°C. Digest the cells and resuspend the cells in a serum-free medium to a density of 1x106. Take 200ul of the cell suspension and apply it to the tiny chamber, contain 500ul of 15% serum-containing medium (without bubbles) in the bottom chamber, and incubate in a 37°C, 5% CO2 incubator for 48 hours.
Apoptosis Analysis
The cells were tagged with AnnexinV-FITC and Propidiumiodide (Solarbio, Beijing, China), and apoptosis was detected by flow cytometry. MDA-MB-231 cells were installed under a thickness for 1x106 cells, and cultured in a 37°C, 5% CO2 cell hatchery. After 24h, they were treated with drugs for 48h. Collect cells (1X106) and wash with pre-cooled PBS. Resuspend the cells with 1ml 1X Binding Buffer to make the cell density attain 1X106 cells/ml. Add 100ul cells to each tube, apply 5ul Annexin V-FITC to the tube and protect from light at room temperature for 10min. Then add 5ul PI and incubate for 5min at room temperature in the dim. Add PBS to 500ul.
Cell Cycle Analysis
The cells were tagged with Propidiumiodide (Solarbio, Beijing, China), and the cell cycle was detected by flow cytometry. MDA-MB-231 cells were transplanted into a culture dish with a density of 1x106 cells/well, and cultured in a 37℃, 5% CO2 cell hatchery. After 48 hours of drug treatment, the cells were collected. Add 1ml 70% ethanol to fix for 2h to overnight, store at 4℃. Add 100ul RNase A, 37℃ water bath for 30min. Then pour 400ul PI dye solution and shelter from light at 4℃ for 30min.
qPCR
Use TRIzol Universal Reagent (TIANGEN BIOTECH, Beijing, China) to extract total RNA. Transcribed into cDNA using RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Shanghai, China). Using ChamQ Universial SYBR qPCR Master Mix Kit (Vazyme, Nanjing. China), real-time quantitative PCR was performed in a fluorescence quantitative PCR instrument chemiluminescence imaging system (Bio-Rad, Harkles, CA, USA). The GAPDH gene have been utilized concerning illustration a reference gene, furthermore estimated mRNA levels were ascertained utilizing those 2-△△Ct method.
Western Blotting
Western blot have been utilized similarly as method should recognize total protein statement. Tissues and cells were lysed in a blended cushion comprising about RIPA:PMSF (100:1). The extracted protein was abrupted by 10%SDS-PAGE and converted to polyvinylide fluoride (PVDF) membranes. The membranes and the primary antibody were incubated overnight to a shaker in 4°C. The film have been washed 3 times for Tris-HCl solution+Tween-20 (TBST). The film were incubated for CoraLite594-conjugated Goat Anti-Rabbit IgG(H+L) secondary antibody for 2h toward room temperature for a shaker. The western blot was detected by the ultra-sensitive ECL chemiluminescence method, and the band intensity was monitored by the chemiluminescence imaging system(Bio-Rad, Harkles, CA, USA).
In Vivo Proliferation Assays
For in vivo proliferation assays,1×107cells/100ul PBS were subcutaneously administered into the flanks of male BALB/c-nu mice(6-8w,n=3)(Liaoning Changsheng Biotechnology, Liaoning, China). Mice were sacrificed after 7w, and tumor volumes (long × width2/2)were measured. All animal test was sanctioned by the for the Articles of Association of the Experiments Animal Ethics and Welfare Committee of Jinzhou Medical University.
Hemoxyeosin Staining (HE)
BALB/c-nu mice cancer tissue sections were deparaffinized and hydrated. Hematoxylin stained for 2min. Stain with eosin for 1min and wash with water for 3min. The tissue sections are dehydrated and transparent. Adhere to the sheet with neutral gum. Observe the staining results with a slide scanner.
Immunohistochemistry (IHC)
Dewax and hydrate the paraffin-embedded sample. In sodium citrate buffer (10mmol/L, pH6.0), high pressure boiled at 100°C to extract the antigen. 0.3% hydrogen peroxide to restrain endogenous peroxidase. Blocked for goat serum during room temperature to 15min. The areas were incubated for rabbit anti-Caspase9 (1:200, 10380-1-AP, Proteintech, Wuhan, China), CyclinD1 (1:200, 26939-1-AP, Proteintech, Wuhan, China) monoclonal antibodies overnigh. The areas were incubated for biotin-labeled goat anti-rabbit IgG to 15min at room temperature. Drop an appropriate amount of horseradish enzyme labeled streptavidin working solution and incubate at room temperature for 15min. DAB staining, hematoxylin counterstain, then dehydrated, transparent, and eventually wrapped with neutral gum.
TUNEL Assays
Tissue sections were labeled with TdT-mediated dUTP Nick-End Labeling (TUNEL, TransGen Biotech, Beijing, China), and positive expression was observed by an inverted fluorescence microscope(Leica, Germany). 4um sections of paraffin cancer tissues were deparaffinized. Add 100ul cell permeabilization solution (1X PBS comprising 0.1% Triton X-100) to the tissue surface. Mix 50ul 1x Labeling Solution with 2ul TdT and apply dropwise to the surface of the slice, and label for 1h at 37℃ in the dark. Perform cell permeability three more times. Use mounting tablets containing DAPI for mounting.
Statistical Analysis
All data were passed by GraphPad Prism v.9.0.0 measurable examination. The analysis might have been performed utilizing one-way ANOVA and two-way ANOVA. Information need introduced as a mean ± SD from in any event three autonomous investigations. P<0. 05 might have been regarded statistically noteworthy.