Dulbecco s modified eagle medium dmem with high glucose

The PTMScan® Phospho-Tyrosine Rabbit mAb (P-Tyr-1000) Kit, AMPK, phospho-AMPK at T172, CAMKK2, PTK2, phospho-PTK2 at Y925, phospho STAT3 at Y705, C-Jun, phospho C-Jun at T73 antibodies were obtained from Cell Signaling Technology (Danvers, United States). Anti-β actin–HRP conjugated antibody was purchased from Sigma-Aldrich, United States; STO-609 (7-oxo-7H-benzimidazo [2,1-a]benz[de] isoquinoline-3-carboxylic acid–acetic acid), a CAMKK2 inhibitor, was purchased from Santa Cruz Biotechnology, Inc. (Texas, United States). TPCK-treated trypsin was obtained from Worthington Biochemical Corp. (Lakewood, NJ). Dulbecco’s Modified Eagle Medium (DMEM) with high glucose, fetal bovine serum (FBS), and antibiotic–antimitotic solution were purchased from Gibco (Thermo Fisher Scientific, United States). All other consumables used in the study were from Sigma Aldrich, United States.

The rats were euthanized by intraperitoneal injection of sodium pentobarbital (150 mg/kg). Fibroblasts were isolated from three tissue types: Skin (ear), muscle (hind limb), and heart (ventricle). Tissues of the same type from the three rats were mixed together. The tissues were cut, washed in phosphate-buffered saline (PBS), and minced in a 250 μL drop of 0.025% collagenase type I (Sigma-Aldrich, MO, USA) in a 100 cm tissue culture plate. The small pieces of tissue were transferred into a 50 mL sterile conical tube (Falcon^®, Corning, NY, USA) containing 2.5 mL of 0.025% collagenase type I and incubated at 37°C for 20 min for the skin tissue, 35 min for the muscle tissue, and 50 min for the heart tissue. Then, 5 mL of 0.25% trypsin-EDTA (ThermoFisher Scientific, MA, USA) were added and incubated at 37°C for 10 min. Thereafter, 5 mL of PBS was added, and the tissues were vortexed briefly before being centrifuged at 1000×g for 5 min. Then, the sediment was resuspended with 5 mL of culture media containing Dulbecco’s Modified Eagle Medium (DMEM) with high glucose (Thermo Fisher Scientific, MA, USA), 10% fetal bovine serum (FBS) (Sigma-Aldrich, MO, USA), and antibiotic-antimycotic solution (Thermo Fisher Scientific, MA, USA). The cells were introduced into a 10-cm cell culture dish and incubated for 2 days at 37°C in a 5% CO₂ incubator (Heal Force, Shanghai, China).

Hepatoma cells (HepG2), colon cancer cells (DLD-1), and breast cancer cells (MCF-7) were provided by the Second Hospital of Harbin (China). All cell lines were mycoplasma-free and were authenticated using short tandem repeat (STR) genotyping within the last three years and were verified as being identical to STR profiles in comparable databases (Zhejiang Ruyao Biotechnology Science and Technology Company Limited). Dulbecco’s modified eagle medium (DMEM) with high glucose was sourced from Gibco (USA), Roswell Park Memorial Institute (RPMI) 1640 medium from BioReagent, and fetal bovine serum (FBS) from Invitrogen (USA). Penicillin-streptomycin and phosphate buffered saline (PBS) were purchased from Sigma and Gibco, respectively. Trypsin (0.25%) was obtained from HyClone (USA), and 96-well tissue culture plates from Corning (USA). Cell counting kit-8 (CCK-8) and cell cycle assay kits were purchased from Dojindo Laboratories (Japan) and Shanghai Ruian Biological Engineering Co. Ltd., respectively. AnnexinV-fluorescein isothiocyanate (FITC) and propidium iodide (PI) apoptosis assay kits were obtained from Beyotime Biotechnology. The Q Exactive and Ultimate 3000 UPLC systems were sourced from Thermo Fisher Scientific.

Soya Phosphatidylcholine (SPC; Lipoid S-100; 94% purity), Hydrogenated Soya Phosphatidylcholine (HSPC; Phospholipon, 80% purity) and Dimyristoly Phosphatidylcholine (DMPC; Lipoid-PC, 98% purity) were purchased from Lipoid, Steinhausen, Switzerland. Paclitaxel (PTX) was obtained from ChemieTek, Indianapolis, USA). Analar grade methanol, acetonitrile, absolute ethanol deionized water (DW) and milipore filter (10 kDa) were purchased from Fisher Scientific Ltd., UK. Cholesterol and Fetal bovine serum (FBS) was supplied by Sigma-Aldrich, UK. Lactose Monohydrate (LMH), Microcrystalline Cellulose (MCC; Avicel PH 12), and Starch (Starch-150) were purchased from VWR (BDH Prolab), UK. The Vibrating mesh (Omron NE U22) nebulizer was supplied by Omron Healthcare UK Ltd., UK. The Ultrasonic nebulizer was purchased from Uniclife Healthcare LTD, UK. Dulbecco’s Modified Eagle Medium (DMEM) with high glucose, TryplTrypLE™ Express Enzyme (1X), with phenol red, and AlamarBlue™ cell viability reagent were bought from Gibco, Thermofisher, UK. MRC-5 (ECACC 05090501) and MRC-5 SV2 (ECACC 84100401) cell lines were obtained from Public Health England (Salisbury, UK).

PNS were purchased from Xi’an TianBao Guangyuan Biotech. Ltd. (Shanxi Province, China, purity > 95%), and they include notoginsenoside R₁>5.0%, ginsenoside Rg₁>25.0%, ginsenoside Re >2.5%, ginsenoside Rb₁>30.0%, and ginsenoside Rd >5.0% (Pharmacopoeia of the People’s Republic of China (2020)). A Dulbecco’s Modified Eagle Medium (DMEM) with high glucose was brought from Gibco (United States, 1980922). The ATP1A1 inhibitor (digitonin, ST1272) was brought from Biotech (Beijing, China). Sorafenib (SC0236) was purchased from Beyotime, China. The Servicebio^® RT First Strand cDNA Synthesis Kit (G3330) and 2×SYBR Green qPCR Master Mix (High ROX) (G3322) were obtained from Servicebio. TRIzol and RAPA were used in this study. ATP1A1, ERK, and p-ERK antibodies were purchased from Cell Signaling Technology (United States; batch numbers are 23565S-1, 4695S-14, 9101S-28); mTOR, p-mTOR, AKT, and p-AKT were purchased from Abcam (Abcam, United Kingdom; batch numbers are GR3181969-14, GR112975-29, GR43522-33, and GR297104-1). β-Actin antibody (Wuhan Servicebio Company; lot number: 180926), FITC rabbit antibody (Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.; lot number: 133027), and DAPI (Beijing Beyotime Biotechnology Co., Ltd.; lot number: C1005) were also used.

Four human neuroblastoma cell lines were used in our research. SK-N-BE(2) cells and SH-SY5Y cells were purchased from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences (Shanghai, China). NBL-W-S and SK-N-AS cells were obtained from the State Key Laboratory of Hematology Oncology of the Ministry of Health, Shanghai Children’s Medical Center (Shanghai, China). SK-N-BE(2), SH-SY5Y and NBL-W-S cells were maintained in DMEM (Dulbecco’s Modified Eagle Medium) with high glucose (Life Technologies, Grand Island, NY, USA). SK-N-AS cells were cultured in DMEM with low glucose (Life Technologies, Grand Island, NY, USA). Both media were supplemented with 10% FBS (Life Technologies, Grand Island, NY, USA) and 1% penicillin/streptomycin (Sigma-Aldrich Co, St. Louis, MO). Cells were cultured as monolayer in a humidified atmosphere containing 5% CO₂ at 37°C. When cells reached a confluence of 60-80%, media were switched to serum-free media for drug treatment.