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1

Matrine Inhibits AML Cell Viability

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Human AML THP-1 cells were purchased from the Cell Bank of Chinese Academy of Sciences. Matrine was purchased from Tianyuan Biological Agent Plant. The Cell Counting Kit-8 (CCK-8) was purchased from Dojindo Molecular Technologies, Inc. RPMI-1640 medium, fetal bovine serum (FBS), dimethyl sulfoxide dissolving (DMSO), penicillin and streptomycin were purchased from Invitrogen; Thermo Fisher Scientific, Inc. The ELISA reader ELx800 was obtained from Bio-Tek Instruments, Inc. Inverted phase contrast microscope was obtained from Olympus Corporation. LY294002 was purchased from Cell Signaling Technology, Inc. and was dissolved in DMSO according to the manufacturer's instructions. Antibodies, including anti-PI3K, anti-phosphorylated (p)-PI3K, anti-mTOR, anti-p-mTOR, anti-Akt, anti-p-Akt and anti-β-actin were purchased from ABclonal Biotech Co., Ltd. The current study was approved by the Ethics Committee of Bengbu Medical College (Bengbu, China).
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2

Quantifying Protein Expression Dynamics

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Total protein was extracted and protein concentration was determined with the BCA Protein Assay Kit (Pierce, Rockford, IL, United States). Equivalent amounts of protein samples were uploaded and separated by SDS-PAGE and then electrotransferred to polyvinylidene difluoride (PVDF) membranes (Millipore Corp, Atlanta, GA, US). The membranes were blocked in 5% non-fat dry milk powder at room temperature for 1 h, and then incubated overnight at 4°C with primary antibodies: anti-FGF3 (1:1000 dilution, Abclonal, Cat # A19052), anti-FGF4 (2 µg/ml, Abclonal, Cat # ab65974), anti-FGF19 (1:1000 dilution, Abcam, Cat # ab85042), anti-EGFR (1:1000 dilution, Abclonal, Cat # A11351), anti-phospho-EGFR-Y1068 pAb (1:1000 dilution, Abclonal, Cat # AP0301), anti-mTOR (1:1000 dilution, Abclonal, Cat # A11354), anti-phospho-mTOR (2448) (1:1000, Abcam, ab109268), anti-p44/42 MAPK (Erk1/2) (1:1000, CST, 4695S), anti-phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (1:2000, CST, 4370S), anti-GAPDH (1:1000 dilution, CST, Cat#5174S), anti-beta-tubulin (1:1000 dilution, CST, Cat#2146). Membranes were then incubated with HRP-conjugated secondary antibodies at room temperature for 1 h. The signals of bands were detected by ECL reagents.
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3

Western Blot Analysis of Autophagy and Cell Signaling Proteins

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Liver tissues or harvested cells were homogenized and sonicated in RIPA (strong) protein extract buffer (Beyotime) containing a protease inhibitor and phosphatase inhibitor (Mannheim Roche, Germany). Equal amounts of protein were separated on SDS-polyacrylamide gels in a mini-gel apparatus (Bio-Rad, Hercules, CA, USA). Next, the protein bands were transferred onto polyvinylidene fluoride (Millipore, IPVH00010, Burlington, MA, USA) membranes that were blocked with 5% milk at room temperature for 1 h. The membranes were then incubated overnight at 4°C with the following primary antibodies: anti-Beclin1 (Abclonal, A7353), anti-ATG3 (Abclonal, A5809), anti-Caspase-3 (Abclonal, A19654), anti-mTOR (Abclonal, A2445), anti-AMPK (Abclonal, A16960), anti-LC3-II&LC3-I (CST, #12741S), anti-p-AMPKα1 (CST, #2537S) or anti-β-actin-HRP (Bioworld, BS6007MH). After being washed 3 times, the membranes were incubated with goat anti-rabbit antibody (Abclonal, AS014) for 1 h at room temperature. Next, the membranes were washed again, and the immunostained protein bands were visualized by enhanced chemiluminescence (Thermo Scientific, 32106, Waltham, MA, USA). NoveVR 4. 6. 2 software (Bio-Rad) was to analyze the staining intensity of each band.
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