The lentiviral vector was prepared as described previously.23 ,24 (link) The NF-κB sensing and IL-4 secreting pCDH-NFKBRE-mIL4-copGFP vector12 (link) or the control pCDH-CMV-MCS-copGFP vector (System Biosciences, Palo Alto, CA), pMD2G VSV-G envelope vector, and psPAX2 packaging vector were cotransfected to human embryonic kidney 293T cells (ATCC, Manassas, VA) using the calcium phosphate transfection kit (Clontech, Mountain View, CA) and 25 μM chloroquine. After dilution in the MSC culture medium supplied with 6 μg/mL of Polybrene (Sigma-Aldrich, St. Louis, MO), the virus was infected into mouse MSCs at multiplicity of infection (MOI) of 40. The infectious efficiency of this method was previously confirmed.23 ,24 (link)
Human embryonic kidney 293t cells
Manufactured by American Type Culture Collection
Sourced in United States
Human embryonic kidney 293T cells are a cell line derived from human embryonic kidney cells. They are widely used in cell biology research and biotechnology applications.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (8 mentions)
Penicillin, by Thermo Fisher Scientific (4 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (3 mentions)
Calcium phosphate transfection kit, by Takara Bio (2 mentions)
Polybrene, by Merck Group (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Rpmi 1640, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Avantor (2 mentions)
Penicillin streptomycin, by Wisent (2 mentions)
Penicillin streptomycin, by Caisson (2 mentions)
Dulbecco modified eagle medium (dmem), by Wisent (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (2 mentions)
Concentrated ammonia aqueous solution, by Sinopharm (2 mentions)
Deionized water, by Merck Group (2 mentions)
Trypsin, by Thermo Fisher Scientific (2 mentions)
Dimethyl sulfoxide (dmso), by Thermo Fisher Scientific (2 mentions)
3 aminopropyltriethoxysilane aptes, by Merck Group (2 mentions)
Anhydrous ethanol, by Sinopharm (2 mentions)
33 protocols using human embryonic kidney 293t cells
1
Lentiviral Transduction of Mouse MSCs
2
Isolation and Culture of Esophageal Cell Types
3
Cell Line Maintenance and Characterization
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The following cell lines were obtained from American Type Culture Collection (ATCC): human embryonic kidney 293T cells (ATCC CRL-11268); African green monkey kidney epithelial Vero cells (ATCC CCL-81); and baby hamster kidney BHK-21 (ATCC CCL-10). These cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (FBS), 2 mM L -glutamine, penicillin (100 U ml−1) and streptomycin (100 U ml−1), and maintained in a 5% CO2 humidified atmosphere at 37 °C. TZM-bl (JC53-bl) cells were obtained from the NIH AIDS Reagent Program, as contributed by John Kappes and Xiaoyun Wu (University of Alabama, Birmingham, AL) and maintained as above. TZM-bl is a HeLa cell clone genetically engineered to express human CD4, CXCR4 and CCR5, and to contain HIV Tat-responsive reporter genes for firefly luciferase (Luc) and β-galactosidase under regulatory control of the HIV-1 long terminal repeat31 (link). MS40L cells32 (link), kindly provided by Xin Luo (Virginia Polytechnic Institute, Blacksburg, VA), were maintained in Iscove's modified Dulbecco's medium plus 10% FCS and antibiotics, and used for B-cell feeder cultures. Expression of human CD40L by MS40L cells was confirmed by indirect immunofluorescence assay. All cell lines tested negative for mycoplasma.
4
Endothelial Cell Culture and Transfection
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Human umbilical vein endothelial cells (HUVECs) were purchased from ScienceCell Research Laboratories (USA) and cultured according to the standard guideline. HUVECs were cultured in endothelial cell medium (1001, ScienceCell Research Laboratories) supplemented with 5% foetal bovine serum, endothelial cell growth supplement and antibiotic solution.
Human embryonic kidney 293T cells were purchased from ATCC (Manassas, VA, USA) and maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% foetal bovine serum (Gibco, North America), 100 U/ml penicillin and 20 U/ml streptomycin. Cells were incubated at 37°C in a humidified chamber containing 5% CO2.
The adenovirus‐encoding IMP3 (ad‐IMP3) was constructed and amplified by Boi‐Link (Shanghai, China). An adenovirus‐encoding red fluorescence protein (ad‐RFP) was used as a negative control. HUVECs were transfected with ad‐IMP3 or ad‐RFP (100 pfu number/cell) for 48 h for the following experiments.
Si‐IMP3 was purchased from GenePharma Company (Shanghai, China); a non‐targeting siRNA (GenePharma, Shanghai, China) was used as a negative control. HUVECs were transfected using Lipofectamine 3000 reagent (Invitrogen, USA) according to the manufacturer's instructions.
Axitinib was resuspended in DMSO and HUVECs treated with 0.5 μg/ml Axitinib.19
Human embryonic kidney 293T cells were purchased from ATCC (Manassas, VA, USA) and maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% foetal bovine serum (Gibco, North America), 100 U/ml penicillin and 20 U/ml streptomycin. Cells were incubated at 37°C in a humidified chamber containing 5% CO2.
The adenovirus‐encoding IMP3 (ad‐IMP3) was constructed and amplified by Boi‐Link (Shanghai, China). An adenovirus‐encoding red fluorescence protein (ad‐RFP) was used as a negative control. HUVECs were transfected with ad‐IMP3 or ad‐RFP (100 pfu number/cell) for 48 h for the following experiments.
Si‐IMP3 was purchased from GenePharma Company (Shanghai, China); a non‐targeting siRNA (GenePharma, Shanghai, China) was used as a negative control. HUVECs were transfected using Lipofectamine 3000 reagent (Invitrogen, USA) according to the manufacturer's instructions.
Axitinib was resuspended in DMSO and HUVECs treated with 0.5 μg/ml Axitinib.
5
Cell Culture Protocols for Human Epithelial Cell Lines
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The human colon epithelium NCM460 cell was purchased from INCELL Corporation (San Antonio, TX). Human embryonic kidney 293 T cells, human colon cancer epithelium Caco2, and HT‐29 cells were obtained from ATCC (Manasses, VA). Cells were grown in Dulbecco's modified Eagle medium (DMEM) in the presence of 10% fetal bovine serum (FBS) and cultured in 5% CO2 at 37°C in a humidified environment.
6
Lentiviral Vector Production and Transduction of Murine MSCs
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As previously described, the lentiviral vector preparation was conducted (Lin et al., 2017 (link); Zhang et al., 2021a (link)). Briefly, we utilized human embryonic kidney 293T cells (ATCC, Manassas, VA, United States) to transfect the control lentivirus vector the mouse IL-4 secreting pCDH-NF-κB-mIL-4-copGFP expressing lentivirus vector combined with psPAX2 packaging vector and pMD2G VSV-G envelope vector, with the calcium phosphate transfection kit (Clontech, Mountain View, CA, United States) and 25 mM chloroquine. Next, the diluted virus was added in MSC culture medium supplemented with 6 mg/ml of polybrene (Sigma-Aldrich, St. Louis, MO, United States) and infected to murine MSCs at multiplicity of infection (MOI) = 100. The genetically modified cells were GPF positive confirmed by fluorescence microscope after 3 days of infection (BZ-X810, Keyence, IL, United States).
7
MDCK and HEK293T Cell Culture
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MDCK cells (ATCC, Manassas, VA, USA) were maintained in minimal essential medium with 5% newborn calf serum. Human embryonic kidney 293T cells (ATCC) were maintained in Dulbecco modified Eagle medium with 10% fetal calf serum. Both cell lines were cultured at 37°C in 5% CO2.
8
Lentiviral Expression of Regulatory Factors
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Self-inactivating lentiviruses expressing green fluorescent protein (GFP), hemagglutinin (HA)-tagged NFI dominant repressor (NFI/EnR), or Drosophila engrailed repressor domain alone (EnR), constitutively active FLAG-tagged NFATc4 (NFATc4-Ala), or constitutively active calcineurin (CaN) were described previously (Wang et al., 2004 (link); Ding et al., 2013 (link), 2016 (link)). These plasmids are available upon request. MEK5-DN lentivirus was also described previously (Doebele et al., 2009 (link)). Human embryonic kidney 293T cells (American Type Culture Collection) were grown in DMEM (Invitrogen) containing 10% heat-inactivated fetal bovine serum (Invitrogen).
9
Osteogenic Differentiation of hBMSCs
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The primary culture of hBMSCs were obtained from ScienCell Research Laboratory (Carlsbad, CA, USA) and cells at passages 3–6 were used for this study. Cells were cultured in α-modified Eagle’s medium (α-MEM; Gibco, Grand island, NY, USA) supplemented with 10% fetal bovine serum (FBS; Gibco) and 1% penicillin/streptomycin (Gibco) as the proliferation medium (PM) as previously described [17 (link)]. Osteoinduction began when hBMSCs reached 70–80% confluence, and the osteogenic medium (OM) contained 10 mM β-glycerophosphate (Sigma-Aldrich, St. Louis, MO, USA), 100 nM dexamethasone (Sigma), and 200 μM L-ascorbic acid (Sigma). Human embryonic kidney (293 T) cells were obtained from the American Type Culture Collection (Manassas, VA, USA) and cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% FBS (Gibco) and 1% penicillin/streptomycin (Gibco). All cells were cultured in a humid atmosphere with 95% air, 5% CO2 at 37 °C.
10
Quantifying APOBEC3G Degradation by HIV-1 Vif
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One hundred nanograms of FLAG-A3G-pcDNA or HA-A3G-pcDNA variants were cotransfected with 200 ng of either Vif-pcDNA or pcDNA3.1(+) empty vector into human embryonic kidney 293T cells (American Type Culture Collection) in 12-well plates (CELLTREAT) using the X-tremeGENE 9 DNA Transfection Reagent (Roche). Cells were incubated at 37°C in 5.0% CO2. At 24 hours after transfection, 2 μM N-carbobenzyloxy-l -leucyl-l -leucyl-l -leucinal (Sigma-Aldrich) or dimethyl sulfoxide control was added. At 48 hours after transfection, the cells were washed once with phosphate-buffered saline and lysed in radioimmunoprecipitation assay buffer with 1× cOmplete Protease Inhibitor (Roche). The lysates were then subjected to Western blot with anti-FLAG M2 monoclonal antibody (mAb) from mouse (Sigma-Aldrich), anti–α-tubulin mAb from mouse (GeneTex), and anti-Vif mAb from mouse (National Institutes of Health AIDS Reagent Program #319) as primary antibodies. Cy3-labeled goat anti-mouse mAb (GE Healthcare) was used as a secondary antibody to detect the signal. The fluorescent signal was detected and visualized with Typhoon RGB Biomolecular Imager (GE Healthcare).
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