For transient transfection, miR-150 mimics (50 nM, Qiagen), miR-150 inhibitors (50 nM, Qiagen), or a negative control siRNA were used and transfected into C-33A cells using the Lipofectamine 2000 Transfection Regent (Invitrogen, USA) according to the manufacturer’s instructions.
Lipofectamine 2000 transfection regent
Manufactured by Thermo Fisher Scientific
Sourced in United States
Lipofectamine 2000 Transfection Reagent is a cationic lipid-based reagent designed for efficient transfection of DNA, RNA, and oligonucleotides into eukaryotic cells. It facilitates the delivery of nucleic acid materials into the cells for various research applications.
Automatically generated - may contain errors
Lab products found in correlation
Dual luciferase reporter assay system, by Promega (2 mentions)
Fluorescence microscope, by Olympus (2 mentions)
Con077, by Genechem (1 mentions)
Pmir vector, by Thermo Fisher Scientific (1 mentions)
Dual luciferase reporter assay, by Beyotime (1 mentions)
P smad3, by Santa Cruz Biotechnology (1 mentions)
Smad3, by Santa Cruz Biotechnology (1 mentions)
Smad2, by Santa Cruz Biotechnology (1 mentions)
P smad2, by Santa Cruz Biotechnology (1 mentions)
Recombinant human tgf β1, by R&D Systems (1 mentions)
P jnk, by Santa Cruz Biotechnology (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
Opti mem medium, by Thermo Fisher Scientific (1 mentions)
11 protocols using lipofectamine 2000 transfection regent
1
Transient Transfection of miR-150 in C-33A Cells
2
Construction of MERS-CoV Spike Pseudotyped Lentivirus
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The GFP-expressing lentiviral transfer plasmid was constructed using pLenti6 Gateway Vector kits (Invitrogen) according to the manufacturer’s protocol. The full-length MERS-CoV spike protein-expressing envelope plasmid was constructed by inserting a codon-optimized synthetic DNA sequence (GenScript) into pCAGG plasmid (Addgene, www.addgene.org ). To produce MERS-CoV Spike pseudotyped lentivirus, 293TN cells were co-transfected with packaging plasmids pLP1 and pLP2, the lentiviral transfer plasmid, and the envelope plasmid pCAGG/MERS-CoV Spike using Lipofectamine 2000 transfection regent (Invitrogen). After overnight incubation, the transfection medium was replaced with fresh 10% DMEM. Forty-eight hours later, culture supernatants containing pseudovirus were collected, centrifuged to remove debris, passed through 0.45-μm filter, and stored at −70°C.
3
TFE3 Knockdown in Cell Lines
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Cells were plated in 6-well plates and transfected with short hairpin RNA (shRNA) for human TFE3 (Obio, Y3619 and Y3620) and a non-target shRNA control (Obio, Y007) at a final concentration of 1 μg/well using Lipofectamine 2000 Transfection Regent (Invitrogen) followed by incubation for 48 h. Next, cells were passaged and analyzed for knockdown efficiency 72 h post-transfection. For viral infection, cells were cultured in 12-well plates and transfected with the TFE3 knockdown virus (Genechem, Shanghai, China, 54,157, 54,158 and 54,159) or a negative control virus (Genechem, CON077) using polybrene. 72 h after transfection, cells were infected at a MOI (multiplicity of infection) of 5 per well in 6-well plates. Puromycin was used to establish a stable cell line.
4
Investigating miR-192-5p Regulation of lncRNA WAC-AS1
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The data base known as starBase was used to identify the relationship between miR-192-5p and lncRNA WAC-AS1. The 3’UTR of lncRNA WAC-AS1, containing the miR-192-5p binding site or mutated target site, was synthesized by carrying out a genomic PCR (ELK Biotechnology) and was cloned into pMIR vectors (Ambion, Austin, TX, USA) to construct the reporter vector lncRNA WAC-AS1 wild-type (lncRNA WAC-AS1-WT) or lncRNA WAC-AS1 mutant-type vector (lncRNA WAC-AS1- MUT). Next, 293T cells were transfected with lncRNA WACAS1- WT or lncRNA WAC-AS1-MUT combined with miR-192-5p mimic or mimic control using Lipofectamine 2000 transfection regent (Invitrogen). The transfected cells were incubated for 48 h and dual-luciferase reporter assay (Beyotime Technology, Jiangsu, China) was performed. Firefly luciferase activity of the plasmids was normalized to Renilla luciferase activity.
5
FOXO4 3'UTR Luciferase Assay
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Wild type or mutant 3′ untranslated region (UTR) of FOXO4 containing the putative binding sites for miR-150 were subcloned into psiCHECK-2 Vectors. The constructed plasmids were transfected into C-33A cells or cells expressing miR-150 mimics or inhibitors using Lipofectamine 2000 Transfection Regent (Invitrogen). After 48 h, the luciferase activity in cell lysates was evaluated using the Dual-Luciferase Reporter Assay system according to the manufacturer’s instructions (Promega, USA).
6
Regulation of lncRNA WAC-AS1 and miR-192-5p
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Negative control of lncRNA WAC-AS1-shRNA (sh-NC), lncRNA WAC-AS1-shRNA; miR-192-5p inhibitor, negative control of miR-192-5p inhibitor (inhibitor-NC); miR-192-5p mimics, negative control of miR-192-5p mimics (mimics-NC); and negative control of ATG7-over expression plasmid (OE-NC) and ATG7-over expression plasmid (ATG7-OE) were synthesized by Gene Create (Wuhan, China). These synthesized sequences and vectors were then transfected into HepG2.2.15 cells using Lipofectamine 2000 Transfection Regent (Invitrogen; 11668019) according to the manufacturer’s protocol. The cells were collected 24 h after transfection, and qRT-PCR analysis and Western blotting were performed to evaluate gene expression and protein expression, respectively.
7
Establishing Stable Cell Lines with miR-150
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Lentiviral vectors were used to establish stable cell lines expressing miR-150 mimics or inhibitors. Psin-EF2-miR-150-IRES-GFP-puro expressing miR-150 pre-miRNA was constructed using specific primers, 5′-GCCGAATTCGCATAGGGTGGAGTGGGTGT-3′ (forward) and 5′-GCCGGATCCATAGAAACAGGTGTACTTTG-3′ (reverse). The paired miR-150 inhibitors (forward: 5′-GATCCCCCACTGGTACAAGGGTTGGGAGA-3′, reverse: 5′-AATTCTAAAAATCTCCCAACCCTTGTACCAGTG-3′) were inserted into pshRNA-copGFP Lentivectors. 293T cells were used to generate lentivirus containing the sequences of miR-150 precursor or inhibitors. 293T cells were co-transfected with pMD2.G, pSPAX2, and pshRNA-copGFP-inhibitor or Psin-EF2-miR-150-IRES-GFP-puro using Lipofectamine 2000 Transfection Regent (Invitrogen). After 48 h, supernatant was collected and filtered using 0.22 um filter. Filtered supernatant containing lentivirus was next used for transduction. C-33A cells were infected with lentivirus for 24 h and cultured with MEM medium including puromycin (5 ng/mL). Expand cells were then used for further experiments and transduction efficiency was measured by a fluorescence microscope (Olympus, Japan). A control cell line transducted with empty vectors was also established after the same procedure.
8
Investigating TGF-β1 Signaling Pathways
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LPS (Sigma; Escherichia coli serotype 055:B5), recombinant human TGF-β1 (R&D Systems) were used in this study. The antibodies used for the western blot analysis, and are p-JNK (catalog no. sc-81502), JNK (catalog no. sc-7345), p38 (catalog no. sc-398305), p-p38 (catalog no. sc-17852-r), p-Smad2 (catalog no. sc-101801), Smad2 (catalog no. sc-39312), p-Smad3 (catalog no. sc-101154), and Smad3 (catalog no. sc-130218), all purchased from Santa Cruz Biotechnology, Inc. NF-κB inducible reporter plasmid were purchased from InvivoGen (cat no: pnifty2-luc, San Diogo, CA). Lipofectamine 2000 transfection regent was purchased from Invitrogen.
9
HEK293T Cell Transfection Protocol
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HEK293T cells were seeded in 6-well plates (2.0 × 105 cells/well) and transfected the following day using the Lipofectamine2000™ transfection regent (Invitrogen) in serum-free OPTI-MEM medium (Invitrogen), according to manufacturer’s instructions.
10
Validating miRNA-370 binding to KDR UTR
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Wild type or mutant 3′ untranslated region (UTR) of KDR containing the putative binding sites for miRNA-370 were subcloned into psiCHECK-2 Vectors. The constructed plasmids were transfected into HRCEC cells as well as cells expressing miR-370 or inhibitors using Lipofectamine 2000 Transfection Regent (Invitrogen). After 48 h, the luciferase activity in cell lysates was evaluated using the Dual-Luciferase Reporter Assay system according to the manufacturer's instructions (Promega, USA).
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