HTR-8/SVneo cells were generously provided by Professor Yanling Wang (Institute of Zoology, Chinese Academy of Sciences, Beijing, China). The HTR-8/SVneo cell line (https://web.expasy.org/cellosaurus/CVCL_7162 ) was initially developed by Graham et al (24 (link)). The HTR-8/SVneo cell line was generated using freshly isolated evCTB from a first-trimester placenta, which was transfected with a plasmid containing the simian virus 40 large T antigen (SV40). A recent study demonstrated that this cell line contains two distinct populations, one of epithelial origin and one of mesenchymal origin (25 (link)). The trophoblasts were cultured in RPMI-1640 medium (Shanghai Basal Media Technologies Co., Ltd.) supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin and 100 U/ml streptomycin (Biosharp Life Sciences) in humidified air at 37°C with 5% CO2. Fresh medium was replaced every 2 days, depending on the cell status.
Streptomycin
Manufactured by Biosharp
Sourced in China, United States
Streptomycin is a broad-spectrum antibiotic used in laboratory settings. It is a naturally-derived compound produced by the bacterium Streptomyces griseus. Streptomycin functions as an inhibitor of protein synthesis in susceptible microorganisms.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin, by Biosharp (42 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (14 mentions)
Fetal bovine serum (fbs), by Sartorius (5 mentions)
Fetal bovine serum (fbs), by Clark Bioscience (5 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (4 mentions)
Rpmi 1640, by Thermo Fisher Scientific (3 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (3 mentions)
Hyclone, by Cytiva (3 mentions)
Silica gel, by Qingdao Haiyang Chemical (3 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Trypsin, by Biosharp (2 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Biosharp (2 mentions)
Du145, by Thermo Fisher Scientific (2 mentions)
Mcf 7, by Thermo Fisher Scientific (2 mentions)
Minimum essential medium (mem), by Thermo Fisher Scientific (2 mentions)
Rpmi 1640 medium, by BasalMedia (1 mentions)
Lipofectamine 3000 kit, by Thermo Fisher Scientific (1 mentions)
Si nc, by RiboBio (1 mentions)
45 protocols using streptomycin
1
Cultivation of HTR-8/SVneo Trophoblast Cell Line
2
Knockdown of MDK in GBM Cell Lines
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GBM cell lines U251 and U87 were obtained from the Shanghai Institute of Biochemistry and Cell Biology (Shanghai, China). The cells were grown in High-Glucose DMEM containing 15% Fetal Bovine Serum (FBS, Gibco, CA, USA), 100 U/ML Penicillin, and 100 g/ML Streptomycin (Biosharp, CA, USA) at a suitable environment of 37 °C and 5% CO2. To specifically knock down the expression of MDK, the Si-NC and Si-MDK plasmids (sense: GGGAAGGGAAAGGACUAGA
TT; anti-sense: UCUAGUCCUUUCCCUUCCCTT) were purchased from RiboBio (Guangzhou, China) and were transfected to cells with a Lipofectamine3000 Kit (Invitrogen, Waltham, MA, USA) according to the guidance of the manufacturer.
TT; anti-sense: UCUAGUCCUUUCCCUUCCCTT) were purchased from RiboBio (Guangzhou, China) and were transfected to cells with a Lipofectamine3000 Kit (Invitrogen, Waltham, MA, USA) according to the guidance of the manufacturer.
3
Silica Gel Column Chromatography for Biomolecule Separation
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Column chromatography (CC) was performed using silica gel (60–80 mesh, 200–300 mesh, 300–400 mesh, Qingdao Haiyang Chemical Co. Ltd., Qingdao, China). Precoated silica gel 60 F (Merck, Darmstadt, Germany) was used for TLC analysis. Dulbecco’s Modified Eagle Medium (DMEM) and fetal bovine serum (FBS) were obtained from Thermo Fisher Scientific (Carlsbad, CA, USA) and Clark Bioscience (Claymont, DE, USA), respectively. Penicillin and streptomycin were purchased from Biosharp (Hefei, China). Radioimmunoprecipitation assay (RIPA) lysis buffer and the BCA protein assay kit were obtained from Beyotime Biotechnology (Jiangsu, China). Polyvinylidene fluoride (PVDF) membranes were obtained from GE Healthcare (Bensalem, PA, USA). Monoclonal primary antibodies against LOR, FLG, TGM1, GAPDH, p-p38, and p38 were purchased from Cell Signaling Technology (Beverly, CA, USA). All regular solvents and reagents were of reagent grade and were obtained from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA), Acros Organics (Geel, Belgium), and J&K Scientific (Beijing, China).
4
Cell Line Maintenance and Culture
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HEK293T cells, HT1080 cells, BHK-21 cells and THP-1 cells were stored by our laboratory. HEK293T cells were maintained in Dulbecco’s modified Eagle medium (HyClone; Cat No. SH30022.01). HT1080 cells and BHK-21 cells were maintained in minimum Eagle medium (HyClone; Cat No. SH30024.01). THP-1 cells were maintained in RPMI-1640 medium (HyClone; Cat No. SH30809.01). These cells were all supplemented with 10% (vol/vol) fetal bovine serum (FBS, Biological Industries, Lot No. 1719426) and penicillin (100 U/ml)/streptomycin (100 mg/ml) (BioSharp, Cat No. BL505A) at 37°C in a humidified atmosphere containing 5% CO2.
5
Culturing Gastric Cell Lines
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The human gastric carcinoma cell lines SGC7901, BGC823, MKN45, HGC27 and the human gastric mucosal epithelial cell line GES-1 were purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). Cells were cultured in RPMI-1640 (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.), penicillin (100 U/ml), and streptomycin (100 µg/ml; Biosharp, Hefei, China, http://www.biosharp.cn/index.asp ) in a humidified atmosphere containing 5% CO2 at 37°C.
6
Cultivation of Mouse and Human Hepatocytes
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Mouse hepatocyte AML12 cells were bought from the ATCC. They were cultured in Dulbecco’s Modified Eagle Medium/nutrient mixture F-12 (DMEM-F12; Meilun Bio, Dalian, China) with 10% fetal bovine serum, 100 U/ml penicillin/0.1 mg/ml streptomycin (Biosharp, Hefei, China), 1% insulin-transferrin-selenium (Thermo Fisher Scientific, Waltham, MA, USA), and 40 ng/ml dexamethasone (Beyotime). Human hepatocyte MIHA cells, kindly provided by Dr. J. R. Chowdhury (Albert Einstein College of Medicine, New York, NY, USA), were cultured in DMEM (Gibco, Shanghai, China) with 10% fetal bovine serum and 100 U/ml penicillin/0.1 mg/ml streptomycin. All cells were maintained in an incubator at 5% CO2 and 37 °C.
7
NSCLC and Osteoclast Cell Lines Cultivation
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Four kinds of NSCLC cell lines A549, H1355, H1299 and H460, human bronchial epithelial cell line 16HBE, OC precursor RAW264.7 cells and human OB SaOS-2 cells were obtained from ATCC. After thawing, the cells were kept in DMEM high glucose medium (Hyclone Laboratories, San Angelo, TX, USA) containing 10% fetal bovine serum (Tianhang Biological Technology, Huzhou, China), 100 U/mL penicillin (Biosharp, China) and 0.1 mg/mL streptomycin (BioSharp, Hefei, China). The cultivation environment was 37°C and 5% CO2 (link). For the induction of RAW264.7 cells, the Transwell chambers were used. Specifically, 1.104/mL of the transfected A549 cells and 5.103/mL of the RAW264.7 cells were inoculated in the upper and lower chamber, respectively. They were co-cultured in 37°C and 5% CO2 (link) for 7 days, then the differentiation of RAW264.7 cells into OCs was detected. In addition, to elucidate the effect of OPN on bone metabolism through Notch pathway, RAW264.7 cells and SaOS-2 cells were treated with Notch pathway inhibitor LY3039478 (final concentration was 1 nM).
8
Glucose-mediated HepG2 Cell Response
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The HepG2 cell line was provided by the China Center for Type Culture Collection (China), and was maintained in Dulbecco's Modified Eagle's Medium (DMEM; Hyclone, USA) supplemented with a normal concentration of glucose (5.5 mmol/l D-glucose), 10% fetal bovine serum (FBS; Biological Industries, Israel), 100 μg/ml streptomycin, and 100 U/ml penicillin (Biosharp, China) at 37°C in a humidified atmosphere containing 5% CO2. At 70–80% confluency, the cells were cultured in medium without FBS for 12 h, and then treated with a high concentration of glucose (25 mmol/l D-glucose) for 6, 12, and 24 h, after incubation under normal glucose for 18, 12, and 0 h, respectively as previously described (total incubation of 24 h) (18 (link)). The SIKs inhibitor HG-9-91-01 was purchased from MedChemExpress (China).
9
Cell Culture and Transient Transfection Protocol
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All the cell lines used in the present study were obtained from National Collection of Authenticated Cell Cultures (Shanghai, China) and authenticated using STR profiling. The human HEK293T (SCSP-502), and Daoy cells (SCSP-509) were maintained in high glucose DMEM (Gibco, 11995065) supplemented with 10% fetal bovine serum (FBS, Gibco, 10091148), 100 μ/ml penicillin and 100 μg/ml streptomycin (Biosharp, BL505A). The mouse C3H10T1/2 cells (SCSP-506) and MEF were cultured in MEM (Gibco, 11140050) supplemented with 10% FBS and 100 μ/ml penicillin and 100 μg/ml streptomycin. All the cells were maintained in a humidified incubator containing 5% CO2 and 95% air at 37 °C. All the cells were periodically tested for free mycoplasma contamination via PCR. Transient transfection was performed using Lipofectamine 2000 reagent (Thermo Fisher Scientific, 11668019) as per the manufacturer’s instructions.
10
Inducing Mesenchymal Transformation in TC-1 Cells
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The TC-1 cell line was bought from the iCell Bioscience, Inc. (Shanghai, China), and cultured in RPMI 1640 containing 10% fetal bovine serum (Clark Bioscience, Claymont, USA), penicillin (100 kU/L), and streptomycin (100 mg/L) (Biosharp, Hefei, China) at 37°C in a 5% CO2 humidified incubator. When the cells were in good condition, in accordance with the previous study (Zeng et al., 2021 (link)), TGF-β1 (10 ng/ml) was added to induce TC-1 cells transformation into mesenchymal-like cells.
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