siRNA targeting RP11-20G13.3 was designed and synthesized by GenePharma (Shanghai, China). The sequences of the three siRNAs targeting to RP11-20G13.3 were shown in Supplementary Table S3 . Based on the manufacturer’s instructions, SW872 cells were transfected with siRNA using Lipofectamine 2000 (Invitrogen, USA). Briefly, siRNA (125 pmol) and Lipofectamine 2000 (6.67 μl) in Opti-MEM I (250 ml) were dispensed onto 105 cells and incubated at 37 °C in 5% CO2 for 4–6 h. The siRNA-Lipofectamine 2000 mixture was changed with fresh complete medium and further incubated for 24 h. Then medium was changed with oleic acid (0.6 mM, Sigma, USA) in DMEM/F12 1:1 to induce preadipocytes differentiation. The knockdown efficiency was evaluated by qRT-PCR analysis.
Lipofectamine 2000
Manufactured by Merck Group
Sourced in United States, China, Germany
Lipofectamine 2000 is a cationic lipid-based transfection reagent used for the delivery of DNA, RNA, and other nucleic acids into eukaryotic cells. It facilitates the formation of positively charged complexes with negatively charged nucleic acids, which can then be taken up by cells.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (26 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (11 mentions)
Puromycin, by Merck Group (6 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (6 mentions)
Polybrene, by Merck Group (5 mentions)
Poly 1 c, by Merck Group (4 mentions)
Fetal bovine serum (fbs), by Merck Group (4 mentions)
Trizol, by Merck Group (3 mentions)
Hygromycin b, by Merck Group (3 mentions)
Pcmvp neo, by Merck Group (3 mentions)
Sirna, by GenePharma (3 mentions)
Fugene hd, by Promega (3 mentions)
Pcdna3 vector, by Sangon (3 mentions)
Lipofectamine 2000, by Promega (3 mentions)
Rpmi 1640, by Thermo Fisher Scientific (3 mentions)
Lsm 710 confocal microscope, by Zeiss (3 mentions)
Topflash, by Merck Group (3 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (2 mentions)
Psilencer 2.1 u6 hygro vector system, by Thermo Fisher Scientific (2 mentions)
Pcmvp neo x system, by Takara Bio (2 mentions)
169 protocols using lipofectamine 2000
1
RP11-20G13.3 siRNA Knockdown and Adipocyte Differentiation
2
HCC Cell Line Hep3B2.1-7 Functional Experiments
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The human HCC cell line Hep3B2.1–7 was purchased from American Type Culture Collection Co (China). The cells were cultured at 37 °C in 5% CO2 in Eagle’s minimum essential medium supplemented with 10% fetal bovine serum, 100 units/mL penicillin, and 100 μg/mL streptomycin, based on the manufacturer’s instruction. As previously reported [23 (link)], the cells were treated with either sorafenib or lenvatinib at 3 μM for 4 days.
For functional experiments, a specific has-miR-548ah mimic/inhibitor (Ambion) (50 nmol/L) and a corresponding negative control (Ambion) were transfected into the Hep3B2.1–7 cells. The transfection was achieved using Lipofectamine 2000 (Sigma-Aldrich, Beijing, China).
For functional experiments, a specific has-miR-548ah mimic/inhibitor (Ambion) (50 nmol/L) and a corresponding negative control (Ambion) were transfected into the Hep3B2.1–7 cells. The transfection was achieved using Lipofectamine 2000 (Sigma-Aldrich, Beijing, China).
3
Investigating HEV ORF3 Protein Expression in Liver Cell Lines
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THP1 monocytes (ATCC, Manassas, VA, USA) and HepG2 cells (Cell Bank of the Chinese Academy of Sciences, Shanghai, China), authenticated by STR profiling (Supplement S1 ,S2 ), were cultured in RPMI 1640 supplemented with 10% fetal bovine serum (FBS, Gibco). Huh7 and normal hepatocytes LO2 cell lines (stored at Chongqing Key Laboratory of Infectious Diseases and Parasitic Diseases) were cultured in Dulbecco’s Modified Eagle Medium (DMEM, Gibco) containing 10% FBS. Adenovirus vector (Ad-Hu5) and plasmid vectors (pcDNA3.1-GFP) overexpressing HEV ORF3 gene (genotype 1 strain Sar55) were constructed in this study. THP1 macrophages differentiated from monocytes by using 10 ng/mL phorbol12-myristate13-acetate (PMA; Sigma) were transduced with recombinant adenovirus vector expressing ORF3 protein (Ad-ORF3) or control vector (Ad-Hu5) at a MOI of 20. Plasmids premixed with Lipofectamine 2000 (Sigma) were transfected into LO2 cells. Lipopolysaccharide (LPS; Sigma) (100 ng/mL) was added to cells to induce expression of TLR7. Cells were divided into different groups based on treatments with agonists and inhibitors.
4
Constructing Chanti-MACC-1 Monoclonal Antibody
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The mouse anti-human MACC-1 monoclonal antibody was constructed using a conventional approach. The chimeric antibody (Chanti)-MACC-1 was constructed as previously described (21 (link)) The single chain variable fragments of the MACC-1 monoclonal antibody were cloned and inserted into the Pklight vector (Hengfei Bioscience, Inc., Shanghai China). The constant domain heavy chain (CH)-Fc and light chain (CL) fragments were subcloned into the Pklight-anti-MACC-1 vector, which were subcloned into the Peedual 12.4 vector (Hengfei Bioscience, Inc.). Subsequently, this was transfected into CHO-K1SV cells using Lipofectamine® 2000 (Sigma-Aldrich; Merck KGaA) at room temperature for 20 min. The production of Chanti-MACC-1extracted from the CHO-K1SV cells was confirmed by using SDS-PAGE western blotting.
5
Cloning and Stable Expression of NHE6
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peGFP-N3-NHE6 was generously provided by Hiroshi Kanazawa (Osaka University, Japan). pcDNA3-HA/NHE6 was constructed from peGFP-N3-NHE6. For this, the entire NHE6 coding sequence was excised with BamHI and EcoRI restriction enzymes and cloned in pcDNA3-HA vector. pcDNA3-HA/NHE6527–588 was designed from pcDNA3-HA/NHE6 using the following primers: forward, 5′-ATGCGGATCCACCAAAGCAGAGAGTGCTTG-3′ and reverse, 5′-GCATGAATTCTTAATCATCATCTTTCAACTGTT-3′.
HT-1080 cells were stably transfected with polyethylenimine (MirusBio) and positive cells were selected with Geneticin (G418) at 400 μg ml−1. In the case of MDA-MB-231 cells, plasmids were transfected using Lipofectamine 2000 (Sigma-Aldrich) and G418 was added to cell cultures at a concentration of 2 mg ml−1.
HT-1080 cells were stably transfected with polyethylenimine (MirusBio) and positive cells were selected with Geneticin (G418) at 400 μg ml−1. In the case of MDA-MB-231 cells, plasmids were transfected using Lipofectamine 2000 (Sigma-Aldrich) and G418 was added to cell cultures at a concentration of 2 mg ml−1.
6
Optimizing Transfection Efficiency for Gene Delivery
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Materials used in this study were l -histidine hydrochloride, l -arginine, stearic acid, and hydrogen peroxide (30% w/v; Sangon Biotech, Shanghai, People’s Republic of China); polyethylenimine (PEI, branched, molecular weight 25 kDa), Lipofectamine™ 2000, l -buthionine-sulfoximine, 4′,6-diamidino-2-phenylindole dihydrochloride, l -cysteine hydrochloride monohydrate, ethidium bromide, dithiothreitol (DTT, Sigma-Aldrich, St Louis, MO, USA); a luciferase assay kit (Promega, Madison, WI, USA); pDNA (pGL3 and pEGFP) (Shanghai Innovation Biotechnology Co Ltd, Shanghai, People’s Republic of China); enhanced bicinchoninic acid protein assay kit (Beyotime, Nanjing, People’s Republic of China); Dulbecco’s Modified Eagle’s Medium (DMEM), fetal bovine serum, YOYO-1 (Y3601) and penicillin–streptomycin solution 5 kU/mL (Life Technologies, Carlsbad, CA, USA); a Cell Counting Kit-8 (CCK-8, Dojindo Molecular Technologies Inc, Nanjing, People’s Republic of China); and HEK293 cells and HeLa cells (Cell Culture Center of the Shanghai Institutes for Biological Sciences of the Chinese Academy of Sciences, Shanghai, People’s Republic of China). All other reagents were of analytical grade. All animal experiments were performed in accordance with the ethics and regulations of animal experiments of Second Military Medical University (Shanghai, People’s Republic of China).
7
Heparanase Silencing Alters Endothelial Permeability
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Conditionally immortalized mouse glomerular endothelial cells (mGEnC-1) were cultured as described previously [24 (link)]. Heparanase was stably silenced in mGEnC-1 by transfection of a heparanase shRNA construct (Qiagen, Venlo, The Netherlands) with Lipofectamine 2000 into undifferentiated mGEnC-1 and subsequent selection with G418 (Sigma-Aldrich). Differentiated mGEnC-1 were treated with the eNOS inhibitor ADMA (10 μg/ml; Millipore, Amsterdam, The Netherlands) for 18 hours. For transendothelial albumin passage, differentiated mGEnC-1 seeded on polyester membranes in tissue culture inserts (0.4 m pore size; Corning Incorporated, NY, USA) were treated with the eNOS inhibitor ADMA as outlined. Transendothelial albumin passage was determined as described previously [19 (link)].
8
Measuring pNF-κB Luc Reporter Activity
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The pNF-κB Luc reporter plasmid (Beyotime, Shanghai, China), as well as the lipofectamine 2000 (Sigma-Aldrich, California, USA), was transfected into HESCs, followed by being incubated for 48 h. The reporter activity was measured using the luciferase reporter kit (Promega, Wisconsin, USA) via determining the fluorescence intensity using a fluorescent microplate reader (BMG LABTECH, Offenburg, Germany).
9
Endocytosis Inhibition Assay
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Chemical reagents such as 2-(diehtyl amino) ethyl methacrylate (DPA), MTT, mPEG2k, chloroquine, and TRIzol were procured from Sigma Aldrich. Trypsin, fetal bovine serum, Dulbecco's modified Eagle's medium, Lipofectamine 2000, endocytosis inhibitors, and other biological reagents were provided by Sigma-Aldrich.
10
Transfection and Stimulation of Cells
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