Cell Culture and Material
All cell lines were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). The human breast cancer cell lines MDA-MB-231 was cultured in DMEM/F12 (1:1) supplemented with 10% fetal bovine serum (FBS, Biological Industries) and 100 units/ml penicillin-streptomycin (Biological Industries), BT549, HCC1806 and HCC1937 were cultured in RPMI 1640 supplemented with 10% FBS, MDA-MB-468 and MCF-7 was cultured in DMEM medium supplemented with 10% FBS and 100 units/ml penicillin/streptomycin. The human immortalized breast epithelial cell lines 184B5 and MCF-10A was maintained in DMEM/F12 (1:1) medium with 5% horse serum, 500ng/ml hydrocortisone, 0.01mg/ml insulin, 20ng/ml epidermal growth factor, 100ng/ml cholera toxin and 100 units/ml penicillin/streptomycin. All of the cells cultured in a humidified incubator at 37°C and 5% CO2.
Cell Viability Assay
Cell viability was determined by the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium-bromide (MTT) assay. Cells were plated in 96-well plates at a density of 2500 cells per well and cultured for indicated condition, followed by addition of 5mg/ml MTT (Solarbio, Beijing, China) and incubation for at least 4h. The absorbance was measured at 570 nm using a SpectraMax M2 spectrophotometer (Molecular Devices, San Jose, USA).
Clone Formation Assay
The cells were seeded in a 6-well plate at a density of 200 per well. The culture medium was replaced every 2 days, and the cells were continuously observed and cultivated for a period of 2 weeks. Upon completion of experiment, the cells were fixed with 4% paraformaldehyde for 30 minutes, followed by staining with 0.1% crystal violet for 20 minutes. The cloning efficiency was determined using the following model: Clonogenic rate (%) = (number of clones / number of cell seeded) ×100%.
Western Blotting Assay and Antibody information
Cell protein were extracted using RIPA buffer and separated by 10% SDS-PAGE and transferred to NC membranes. The membrane was blocked with 5% skim milk for 1 hour, followed by overnight incubation with primary antibodies. After that, membrane was incubated with horseradish peroxidase-conjugated secondary antibodies at room temperature for 1 hour. After each incubation, the membrane was washed 3 times with PBST. Lastly, immunoblotting was performed using the BioRad Chemidoc MP system, and the band grayscale analysis was performed using Image Lab with GAPDH bands as the reference standard. Primary antibodies include GAPDH (cat.#2118S, Cell Signaling, 1:1000), β-actin (cat.#8480, Cell Signaling, 1:1000), p-AMPK(T172) (cat.#50081T, Cell Signaling, 1:1000), AMPKα(cat.#2532S, Cell Signaling, 1:1000), Caspase 3(cat.#14220T, Cell Signaling, 1:1000), cleaved Caspase 3(cat.#9579T, Cell Signaling, 1:1000)
Mouse Xenograft Tumor Models
The animal protocols were reviewed and approved by the Department of Laboratory Animals, Central South University. All methods described in this study were conducted in accordance with the relevant guidelines and regulations as specified by the Department of Laboratory animals, Central South University. Four to six weeks’ old female nude mouse were obtained from SJA Laboratory Animal Co., Ltd (Hunan, China). MDA-MB-231 cells were washed twice with serum-free medium and collected. Subsequently, they were injected subcutaneously into the back of the mice adjacent to the front limb at a volume of 100μL, with injections administered every other day. The mice were randomly divided into different groups and treated with either vehicle or ZSW-4B (2.5 mg/kg/d). When the average tumor size reached about 100mm3, ZSW-4B treatment was given by intraperitoneal injection every other day. Tumor size and mice body weight were measured every two days and tumor volume was calculated simultaneously. At the end of the experiment, all animal were euthanized, and the tumor masses were dissected and weighed. The tumor volumes were calculated using the equation: V (mm3) = a×b2/2, where “a” represents the largest diameter and “b” represents smallest diameter.
Cell Apoptosis Assay
Cells were seeded in a 6-well plate at a density of 2×105 cells per well. After 12 hours of growth, the cells were treated with medium containing ZSW-4B compounds or vehicle for 24 hours. Subsequently, the cells were subjected to triple washing with PBS and then enzymatically dissociated using EDTA-free trypsin to collect the cells. Following resuspension in PBS, the cells were treated with Annexin V and PI. The sample were then incubated at room temperature in a dark environment for 10 minutes. Cell apoptosis was analyzed by measuring the fluorescence using Flow Cytometer (Beckman Coulter, Brea, CA, USA).
Cell-Cycle Analysis
Cells were collected and washed tree times with pre-chilled PBS. Subsequently, the cells were fixed with 70% ethanol at 4°C for 30 minutes. After removing the ethanol, the cells underwent another three washed with PBS. Then, RNase A and PI staining solution were added, and the cells were stained at 37°C for 30 minutes. Finally, the cell samples were analyzed using a Flow Cytometer (Beckman Coulter, Brea, CA, USA).
Establishment of AMPKα1 knockout cell line
The AMPKα1 cell line was established using the CRISPR-Cas9 system. The design of AMPKα1 sgRNA was performed using an online CRISPR design tool (http://crispor.tefor.net/). The sgRNA sequences were cloned into the plentiCRISPR V2 plasmid and co-transfected with PMD2G and PSPAX2 plasmids into 293T cells to package the virus. After 48 hours of transfection, the supernatant containing the virus was collected and filtered, then mixed with fresh medium. The target cells were treated with the virus, and puromycin was used to select the infected cells for three days. Subsequently, the cells were isolated through a colony formation assay. Single clones were validated using immunoblotting. The AMPKα1-sgRNA primer sequences are as follows:
sg1 forward:5’-CACCGGCGTGTCACCCAGAATGTAG-3’
reverse: 5’-AAACCTACATTCTGGGTGACACGCC-3’;
sg2 forward: 5’-CACCGCCGAGAAGCAGAAACACGAC-3’
reverse: 5’- AAACGTCGTGTTTCTGCTTCTCGGC-3’.
Cell Transfection
Transient transfection was performed when the cell confluency reached approximately 50%. TurboFectTM Transfection Reagent was used for the transfection, following the manufacture’s instructions. Subsequent experiments were conducted 24 hours after transfection.
Phosphoproteomics analysis
MDA-MB-231 cell lines was seeded in 10 cm dishes at a density of 2×106 cells per dish and cultured overnight. After overnight incubation, the cells were treated ZWS-4B for a duration of 24 hours. Subsequently, the cells were washed tree times with pre-chilled PBS and lysed using RIPA buffer to extract the total cellular proteins, which were then stored at -80°C for further analysis. The subsequent processing of protein samples, LC-MS/MS identification and bioinformatics analysis were conducted in Zhongke New Life Co. Ltd (Shanghai, China).
Immunohistochemistry staining (IHC).
IHC staining was performed using 4μm paraffin-embedded tumor tissue sections. The primary antibodies against Ki-67 (9449, Cell Signaling Technology, USA), were diluted 1:250 or 1:500, respectively, and then incubated at 4 °C overnight in a humidified container. After three washes with PBS, the tissue slides were treated with a non-biotin horseradish peroxidase detection system according to manufacturer’s instructions (ZSGB-BIO, China) and photographed using an LEICA DM3000 LED (Leica DMshare (v3), Germany). IHC staining was evaluated by two independent pathologists. The Ki67 and ASS1 signals were detected in the nucleus and cytoplasm, respectively. To evaluate Ki67 and ASS1, a semi-quantitative scoring criterion was used in which both the staining intensity and positive areas were recorded. A staining index (values 0–12), which was obtained as the product of the intensity of positive staining (weak, 1; moderate, 2; strong, 3) and the proportion of positive cells of interest (0%, 0; <25%, 1; 26–50%, 2; 51–75%, 3; >76%, 4), was calculated.
Hematoxylin and eosin (HE) staining.
HE staining was conducted according to routine protocols. Briefly, after deparaffinization and rehydration, tissue sections were stained with hematoxylin solution (ZSGB-BIO, China) for 5 min followed by 5 dips in 1% acid ethanol (1% HCl in 75% ethanol) and then rinsed in distilled water. Then the sections were stained with eosin solution (ZSGB-BIO, China) for 3 min and followed by dehydration with graded alcohol and clearing in xylene. The mounted slides were then examined and photographed using an LEICA DM3000 LED (Leica DMshare (v3), Germany).