The human DTL gene (NM_016448) overexpression plasmid pEZ-M03-DTL was constructed by GeneCopoeia (Guangzhou, China). HEK293 cells were purchased and cultured in DMEM (BOSTER, Wuhan, China) containing 1% penicillin/streptomycin (Invitrogen) and 10% fetal bovine serum (Sigma) at 5% CO2and37 °C. For DTL overexpression, cells were transfected with the empty vector or the DTL overexpression plasmid (Ctrl: transfected with empty plasmid, DTL: transfected with DTL-overexpressing plasmid) using Lipofectamine 2000 (Thermo Fisher Scientific) according to the manufacturer's instructions.
Dulbecco modified eagle medium (dmem)
Manufactured by Boster Bio
Sourced in China, United States
DMEM (Dulbecco's Modified Eagle's Medium) is a cell culture medium commonly used for the growth and maintenance of various cell lines. It provides a balanced salt solution with essential nutrients and growth factors required for cell proliferation and survival.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (6 mentions)
Penicillin, by Solarbio (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Streptomycin, by Boster Bio (1 mentions)
Penicillin, by Boster Bio (1 mentions)
Milli q purification system, by Merck Group (1 mentions)
Mueller hinton broth (mhb), by Boster Bio (1 mentions)
Phosphate buffered saline (pbs), by Boster Bio (1 mentions)
Trizma base, by Merck Group (1 mentions)
Agarose, by Lonza (1 mentions)
Szx16 dissecting microscope, by Olympus (1 mentions)
Matrigel, by BD (1 mentions)
Scrambled sirna, by GenePharma (1 mentions)
Penicillin streptomycin solution, by Solarbio (1 mentions)
Cal 27, by Procell (1 mentions)
Scc 25, by Procell (1 mentions)
Fetal bovine serum (fbs), by Boster Bio (1 mentions)
Bca protein assay kit, by Boster Bio (1 mentions)
24 protocols using dulbecco modified eagle medium (dmem)
1
DTL Gene Overexpression in HEK293 Cells
2
Culturing Human Epithelial and Rhabdomyosarcoma Cells
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Human normal gastric epithelial cell (GES-1 cells) and human rhabdomyosarcoma cells (RD) were obtained from Fenyang College of Shanxi Medical University (Fenyang, China) and cultured in DMEM (Boster Biological Technology) supplemented with 10% FBS (Biological Industries Technology), 1% penicillin and 1% streptomycin (Boster Biological Technology). The cells were maintained in an incubator (Thermo Fisher Scientific, Inc.) at 37˚C with 5% CO2.
3
Oligonucleotide Protocol for Molecular Assays
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All the oligonucleotides were obtained from Sangon Biotech (Shanghai, China). The 2000 bp DNA marker was bought from Takara (Tokyo, Japan). Magnesium chloride hexahydrate was purchased from Tian Li (Tianjin, China). Trizma base was obtained from Sigma-Aldrich (St. Louis, MO, USA). Agarose was bought from Lonza (Rockland, ME, USA). PBS, Mueller–Hinton broth (MHB) and DMEM were purchased from Boster (Wuhan, China), Land Bridge (Beijing, China) and Life technologies (Waltham, MA, USA), respectively. The water used in all experiments was prepared via a Millipore Milli-Q purification system with a resistivity higher than 18 MΩ cm-1.
4
Lymphatic Metastasis Model of Cervical Cancer
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Female athymic nude (nu/nu) 4-week-old mice (n=40) were purchased from Beijing HFK Bio-technology Co, Ltd. (Beijing, China). The studies were approved by the Committee on the Ethics of Animal Experiments of Tongji Medical College (Wuhan, China). Mice were maintained in the accredited animal facility of Tongji Medical College at 20–26°C in a 12-h light/dark cycle. A lymphatic metastasis model of cervical cancer was used as previously described (16 (link)). Briefly, 5×106 tumor cells in 50 µl serum-free DMEM (Boster Biological Technology, Co., Ltd., Wuhan, China)/Matrigel (BD Biosciences) at a 9:1 ratio were injected subcutaneously into the claw pads of the mice. Tumor size (mm3) was measured and calculated by the following formula: Volume = (width)2 × length / 2. When primary tumors reached ~150 mm3 in size, metastases were tracked by optical imaging of luciferase activity originating from tumor cells using the IVIS Spectrum system (Caliper Life Sciences; PerkinElmer, Inc., Waltham, MA, USA). Subsequently, when primary tumors reached ~250 mm3 in size, the mice were euthanized, and their popliteal and inguinal lymph nodes were excised. Metastases of tumor cells in the lymph nodes were confirmed by detection of tumor-expressed RFP under a SZX16 dissecting microscope (Olympus Corporation). The incidence of metastasis-positive mice in each group was calculated.
5
Cell Culture and Transfection Protocol
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Cells were cultured in DMEM (Boster, China) with 10% FBS (HyClone, United States) in a humified atmosphere of 95% O2 and 5% CO2. Scrambled siRNAs were purchased from Shanghai GenePharma. Subsequently, cells were transfected with a Lipo3000 kit according to the manufacturer’s instructions.
6
Preparation and Characterization of Titanium and Barium Composite Particles
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Commercial titanium particles (Ti) used in cellular experiments were bought from Pfizer (New York, NY). The size of Ti particle was defined by an electronic scanner microscope and NIH imaging software, and the average diameter was observed to be 4.74 ± 1.82 μm. The particles were rinsed for no endotoxic conglutination, according to the protocol by Ragab et al.24
The Ti particles were disposed in 25% nitric acid for 20 h, followed by incubation in a mixture of 95% ethanol and 0.1 N NaOH at 30°C for 20 h. The procedure was performed twice. Between each step, the Ti was rinsed 3∼5 times with PBS until the used PBS pH was not more than 1 U of the fresh PBS. Then the stock solution was concentrated to 1 mg/ml in DMEM (Boster, China) and 1% antibiotics. Simplex P BC powder (Howmedica Osteonics, Allendale, NJ) was constituted of 10% barium sulfate, 15% PMMA, and 75% methylmethacrylate styrene copolymer. According to the manufacturer the BC particle diameters ranged from < 1 μm to approximately 100 μm. One mg/ml BC mixture was made in 1% antibiotic DMEM.
The Ti particles were disposed in 25% nitric acid for 20 h, followed by incubation in a mixture of 95% ethanol and 0.1 N NaOH at 30°C for 20 h. The procedure was performed twice. Between each step, the Ti was rinsed 3∼5 times with PBS until the used PBS pH was not more than 1 U of the fresh PBS. Then the stock solution was concentrated to 1 mg/ml in DMEM (Boster, China) and 1% antibiotics. Simplex P BC powder (Howmedica Osteonics, Allendale, NJ) was constituted of 10% barium sulfate, 15% PMMA, and 75% methylmethacrylate styrene copolymer. According to the manufacturer the BC particle diameters ranged from < 1 μm to approximately 100 μm. One mg/ml BC mixture was made in 1% antibiotic DMEM.
7
Isolating Rat Cerebral Cortex Cells
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SD rats were treated with ethanol, and the cerebral cortex was isolated. The cortex was digested with trypsin solution, and tissue fragments were dissociated through pipetting digestion was terminated by adding Dulbecco’s modified Eagle’s medium (DMEM) (Bosterbio, Wuhan, China) culture solution containing 10% fetal bovine serum (FBS) (Gibco, Grand Island, USA). Cells were centrifuged and resuspended in a medium, and then cultured at 37 °C with 5% CO2. After 48 hours, the culture medium was replaced regularly. When the primary cells reached 90% confluency, they were digested and transferred to a new culture flask. The nonadherent cells were seeded into another flask, while the adherent cells (microglia) were used for further experiments after 48 hours of culture.
8
Human Oral Squamous Cell Carcinoma Cell Lines
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The human OSCC cell lines CAL-27 and SCC-25 were purchased from Procell Life Science and Technology Co., Ltd. (Hubei, China). The HOK cell line was provided by the School and Hospital of Stomatology at Shanxi Medical University. The CAL-27 cell line was grown in Dulbecco’s Modified Eagle Medium (DMEM, Boster, China), the SCC-25 cell line was grown in DMEM/Nutrient Mixture F-12 (DMEM/F12, Boster, China), and the HOKs were grown in Hyclone1640 medium (Hyclone, USA). The medium contained 10% fetal bovine serum (Gibco, USA) and 1% penicillin-streptomycin solution (Solarbio, China) and was maintained in an incubator with 5% CO2 at 37 °C.
9
Evaluating Compound 5f's Effects on PA-Induced Lipid Dysregulation in HepG2 Cells
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HepG2 cells purchased from Procell (China) were grown in DMEM (Boster, China) supplemented with 10% fetal bovine serum (Minhai, China) and 1% penicillin/streptomycin at 37°C and 5% CO2. After growing HepG2 cells overnight, the cells were treated with 500 μM PA (Solarbio, China) for 24 h. For pharmacological treatments, HepG2 cells were treated with different concentrations of compound 5f simultaneously. The treated cells were subjected to a cell viability and TC/TG content assay. Cell viability was measured by MTT assays. 10 μl MTT (5 mg/ml in PBS, Solarbio, China) solution was added and incubated for 4 hours. Media were removed and replaced with 100 μl of DMSO. The absorbance was measured at 490 nm using a microplate reader (SpectraMax Plus 384, Molecular Devices, USA). EC50 values were calculated. To determine TG/TC contents, the cells were gently washed with phosphate-buffered saline and disrupted by sonication. The contents were normalized to total protein using a BCA protein assay kit (Boster, China) and quantified based on the instructions on the corresponding assay kit (Jiancheng, China).
10
Modulating LCN2 Expression in Colorectal Cancer Cells
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The human CRC cell lines of SW480, SW620, HT-29, and RKO were from the American Type Culture Collection, and 293FT cells were from Life Science (Norway); they were respectively cultured in RPMI 1640 (Gibco, Grand Island, USA) and Dulbecco’s Modified Eagle’s Medium (DMEM, Boster Biological Technology, Co. Ltd, Wuhan, China) supplemented with 10% fetal bovine serum (FBS, Haoyang Bioscience, Tianjin, China) and grown at 37 °C under 5% CO2. SW620 and RKO cells that stably expressed LCN2 (designated as SW620-LCN2 and RKO-LCN2; vector control-OB), and SW480 and HT29 cells with LCN2 knockdown (designated as SW480-sh-LCN2 and HT29-sh-LCN2; vector control-SHB) were established by lentiviral shuttle vector (Detail of vector was in part of vector construction).
Leptomycin B (LMB, Cat. No.431050) was purchased from Merck Millipore (Darmstadt, Germany), and incubated with SW480 for 20nM (10 h), SW620-LCN2 and RKO-LCN2 for 40nM (6 h). Bay11-7082 (B5556, Cat. No. 196870) and JSH-23 (J4455, Cat. No. 749886) were from Sigma Aldrich and added to the culture medium at 50 μM, for 3 h and 50 μM for 1 h, respectively.
Leptomycin B (LMB, Cat. No.431050) was purchased from Merck Millipore (Darmstadt, Germany), and incubated with SW480 for 20nM (10 h), SW620-LCN2 and RKO-LCN2 for 40nM (6 h). Bay11-7082 (B5556, Cat. No. 196870) and JSH-23 (J4455, Cat. No. 749886) were from Sigma Aldrich and added to the culture medium at 50 μM, for 3 h and 50 μM for 1 h, respectively.
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