Cell lines and culture
The AML cell lines used in our study were THP-1, KG-1a, U937, NB4, and HL-60.All of them were available in our laboratory.The cells were cultivated in RPMI-1640 medium contained with 10% FBS and incubated at 37 C under 5% CO2 atmosphere.
Chemicals and reagents
Erianin(HY-N0571), GW6471(HY-15372), LY294002(HY-10108) were obtained from Med Chem Express (MCE, NJ, USA).RPMI-1640 medium was obtained from Gibco (MA, USA).The cell counting kit-8 (HY-K0301) was purchased from Solarbio (Beijing, China).
Cell proliferation and viability assays
The CCK8 assay was uesd to monitor the proliferation rates of cells and the inhibition rates of cells.In brief,1×104 cells/well were seeded in 96-well plates averagely and incubated with Eri on a concentration of 0, 1, 3.3, 10,33,100,333,1000,3333 nm, then added 10µl CCK8 at 0,24,48,72 h to terminate the operation.The Optical density (OD) value detection were performed after incubating for 2h and was recorded at 450nm by microplate reader. 1 × 104 KG-1α or THP-1 cells were totally inoculated in 96-well plates which contain 100 µL medium and then treated with DMSO, the appropriate doses of Eri and GW6471/LY294002 solely or in combination treat for 24 h.Ultimately, the CCK-8 kit was conducted to detect cell viability and then converted to inhibition rates.
Cellular apoptosis detection
To evaluate the cell apoptosis,the particular cells were inoculated on 6-well plates at a density of 3 × 105 cells / well and treated with indicated agentia.After the treatment,the cells were gathered and washed three times with PBS before detection.The apoptosis rate was detected by the Annexin V-FITC Apoptosis Detection Kit (SUNGENE BIOTECH, Tianjing) and the folw cytometric analysis was operated by a CytoFLEX flow cytometer (Beckman Coulter, USA).
Cell cycle analysis
To evaluate the cell cycle analysis, the cells were collected after being treated with Erianin and washed three times with PBS.For the detection, 1 × 106 cells were treated with 75% ethanol and treated overnight at 4℃,then incubated with propidium iodide (PI; 100 mg/ml) for 30 min at room temperature.The BD Biosciences FACSCaliburTM flow cytometer were operating to analyze the cell cycle phases.
Network pharmacology analysis
The 2D structure file of Eri was obtained from Pubchem database and imported into PharmMapper database to predict Eri related potential targets.Using “Acute Myelogenous Leukemia”as keyword to seek for AML-related targets in GeneCards database and utillizing online Venn diagram to receive interactive targets of Eri and AML.Established by STRING database,the protein-protein interaction (PPI) network was ultimately visualized by Cytoscape software to acquire interactive targets.
Molecular docking
As previously described,obtaining the 2D structure of Eri using the Pubchem Database and converting it to 3D form by Openbabel.Next, aquiring the 3D construction of the target from the PDB protein database .The docking and binding energy of the target molecule and Eri were assessed by Autodock Vina.Conducted models by AutoDock Vina software, the results of molecular docking simulation which contained molecular bonds and bond energies for docking were ultimately visualized by Pymol software.
Western Blotting
Western Blotting
After treating with the indicated reagent, the cells were washed three times with PBS and dissolved with PMSF(Beyotime, China) combined with RIPA buffer (Beyotime, China) at a ratio of 1:100.The BCA protein assay kit (Beyotime, China) was used to extract and assesse the concentration of proteins .Briefly, the protein were mixed with 5 × loading buffer and then heated for 10 min.Using Polyacrylamide gel electrophoresis(PAGE) to get protein seperated and then electrotransferred onto a PVDF membrane (Millipore, USA).The membranes were incubated with the indicated primary antibodies at 4°C for 17 h after blocking with a 5% skimmed milk powder.Wait for one night,incubating with relevant secondary antibodies for1h at room temperature, the membranes were ultimately analysed with enhanced chemiluminescence (ECL). The used primary antibodies in our study were as follows :PPARɑ ,PARP,Cleaved-PARP ,Caspase3, Cleaved-Caspase3, P-PI3K, PI3K, AKT,P-AKT,CylinB1,CyclinA2(CellSignalingTechnology,USA);P85ɑ(abcam,China);Bcl-2,Bax ,P27(ZENBIO,China);CDK1(Wanlei,China);GAPDH(Proteintech,USA).
Quantitative real‑time PCR
The TRIzol reagent (Takara, Japan) was uesd to isolated RNA and transcribed them to cDNA by conducting the PrimeScript™ RT reagent kit (Takara).RT-qPCR was operated by CFX Connect™ RT-qPCR system (Bio-Rad, USA) by SYBR® Premix Ex Taq™ II kit (Takara). .
All primers were obtained from Sangon Biotech (Shanghai, China):
The HS-ACTB primers:(Forward:5′-CCTGGCACCCAGCACAAT-3′,Reverse:5′-GGGCCGGACTCGTCATAC-3′);
The PPARɑ
primers:(Forward:5′-AGATTTCGCAATCCATCGGC-3′,Reverse:5′-GCGTGGACTCCGTAATGATA-3′);
The PIK3R1 primers:(Forward:5′-TAGCTCGCGCGATCTAGGGGC-3′,Reverse:5′-CGCGATCAATAAAGCTAG-3′);
The P21 primers:(Forward:5′-AGCGATGGAACTTCGACTTTG-3′,Reverse:5-CGAAGTCACCCTCCAGTGGT-3′);
The P27 primers:(Forward:5′-ATCACAAACCCCTAGAGGGCA-3′,Reverse:5′-GGGTCTGTAGTAGAACTCGGG-3′);
The P53 primers:(Forward:5′-AAAGTGCGTCCGTTCTCAATG-3′,Reverse:5-GGTTCTTCCTCAGAGTACCAAAG-3′)
Statistical analysis
Operating for at least three independent experiments,all data were presented as mean ± standard deviation (SD).Unpaired Student’s t testsone-way or two-way analysis of variance (ANOVA) was perfromed by the GraphPad Prism8 software. P-value less than 0.05 was considered as statistically significant.P-value < 0.05, < 0.01, < 0.001, < 0.0001 respectively represents a statistical significance by “*”,“**”, “***” and “****”.