Human fibroblasts were seeded and transfected with eEF2 silencing RNA (si-eEF2) (#4392420, Ambion) to detect the potential role of eEF2 in tendon healing. Cells were stimulated with 10 ng/mL TNF for 24 h before being transfected with si-eEF2. Cells exposed to silencing RNA for eEF2 and negative controls were transfected for 48 h at a final concentration of 100 nM in each well using Lipofectamine™ 3000 transfection reagent (Thermo Fisher Scientific) and diluted with Opti-MEM reagent.
Opti mem reagent
Manufactured by Thermo Fisher Scientific
Sourced in China, United States
Opti-MEM is a reduced serum medium that supports the growth and maintenance of a variety of cell types in cell culture. It is designed to provide an environment that promotes cell viability and proliferation with reduced serum requirements.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 3000 transfection reagent, by Thermo Fisher Scientific (2 mentions)
Nf κb p65 antibody, by Abcam (1 mentions)
Rabbit anti humanβ actin monoclonal antibody, by Proteintech (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Luciferase assay, by Promega (1 mentions)
Psicheck 2 plasmid, by Promega (1 mentions)
Dual glo luciferase assay kit, by Promega (1 mentions)
Prl sv40 vector, by Promega (1 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
Trypsin, by Abnova (1 mentions)
5 protocols using opti mem reagent
1
Investigating eEF2 Role in Tendon Healing
2
CFD Silencing Impacts Collagen Production
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Cells were seed in 12-well plates and incubated with CFD silencing RNA (siRNA CFD) to detect whether CFD impacts Col1a1 production. Fibroblasts were incubated with a final concentration of 100 nM siRNA CFD mixed with Lipofectamine™ 3000 transfection reagent (Thermo Fisher Scientific), Opti-MEM reagent was used for dilution.
3
Synthesis and Characterization of BrMC
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BrMC was synthesized according to previous described 26 (link). The molecular formula is C16H11BrO4, the character is yellow like powder, the molecular weight is 347, and its purity is 99.5% by HPLC. BrMC was diluted with dimethyl sulfoxide (DMSO) to 10 mmol/L, the desired concentration was diluted to by DMEM medium before application. TGF-β and TNF-α from Sino Biological (Beijing,China). NF-κBP65 inhibitors (SN50) and FoxM1 specific inhibitors, thiostrepton (THI), are purchased from Sigma-Aldrich (St. Louis, MO, USA). NF-κBp65 antibody was purchased from Abcam (Burlingame, CA, USA). The Rabbit anti human β-actin monoclonal antibody was derived from Proteintech (Chicago, USA). The following antibodies were purchased from CST (Massachusetts, USA): Anti FoxM1, CD44, Oct4, CD133 antibodies. The recombinant adenovirus containing pHBad-MCMV-GFP plasmids and pHBad-MCMV-GFP-NF-κBp65 plasmids (virus titer: 1010pfu/mL, 2 mL) was prepared by Han Heng Biotech Corp (Shanghai, China). The FoxM1expressing lentivirus, ENi.s (enhanced infection reagents and Polybrene were purchased from Genechem (Shanghai, China), puromycin purchased from Jikai gene company, Catalog No.REVG0002, Shanghai, China. Opti-MEM reagent is the product of Invitrogen (China, Shanghai).
4
Characterizing RPL29P2 and COL1A1 Regulation
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The promoter of RPL29P2 with the CArG element was amplified by PCR from the HPMCs. The PCR products were ligated (Takara, Japan) into the pRL-SV40 vector (Promega, USA). The HPMCs were transfected with OPTI-MEM reagent (Invitrogen, USA). The transfections were repeated three times. The extraction was prepared for the luciferase assay (Promega) after transfection by an Assay Kit (Promega, USA) (Fig. 3 ).
The 3'-UTR fragments of RPL29P2 or COL1A1 were amplified by PCR from the HPMCs. The products were gel-purified and ligated (Takara, Japan) into the psiCheck-2 plasmid (Promega, USA), named psi-CHECKTM-2- RPL29P2 or COL1A1. The mutated sequences were constructed by a KOD-plus Mutagenesis Kit (). HG- HPMCs were cotransfected with miR-1184 (1 µl, 20 µM), wild-type (wt, 0.5 µg) or mutant (mut, 0.5 µg) RPL29P2 or COL1A1 vector and Lipofectamine 2000 (1 μl, Invitrogen, USA). Luciferase activity was tested using the Dual-Glo luciferase assay kit (Promega, USA).
The 3'-UTR fragments of RPL29P2 or COL1A1 were amplified by PCR from the HPMCs. The products were gel-purified and ligated (Takara, Japan) into the psiCheck-2 plasmid (Promega, USA), named psi-CHECKTM-2- RPL29P2 or COL1A1. The mutated sequences were constructed by a KOD-plus Mutagenesis Kit (). HG- HPMCs were cotransfected with miR-1184 (1 µl, 20 µM), wild-type (wt, 0.5 µg) or mutant (mut, 0.5 µg) RPL29P2 or COL1A1 vector and Lipofectamine 2000 (1 μl, Invitrogen, USA). Luciferase activity was tested using the Dual-Glo luciferase assay kit (Promega, USA).
5
Silencing TRPV4 to Explore KET's Effect
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In order to explore the effect of TRPV4 on hippocampal neuronal cells after KET treatment, we needed to silence the TRPV4 gene. Therefore, we constructed a small interfering RNA expression vector for TRPV4 and transfected into cells. The sequence encoding the shRNA (small hairpin RNA) was then initiated using the Pol III promoter. The sequences of siRNA TRPV4 (siTRPV4) were as follows: sense oligo: 5'-UCAUAUCGGCUU-UCUUGUGAG-3', antisense oligo: 5'-CACAAGAAA-GCCGAUAUGAGG-3'. Next we transfected siTRPV4 into cells using the Lipofectamine TM 3000 (L3000015, ThermoFisher, Waltham, MA, USA). Cells were first digested with 0.25% trypsin (P5259, Abnova, Taibei, Taiwan) for 24 hr. 30 pmol of siTRPVC4 was first mixed with 50 μL of Opti-MEM reagent (31985062, Invitrogen). 3 μL of Lipofectamine TM 3000 reagent was diluted with 50 μL of Opti-MEM reagent. The two mixtures were then mixed and left for 15 min (room temperature). Finally, the above mixtures were added to the cells separately. Cell transfection efficiency was measured by qRT-PCR and western blot after 48 hr. After successful transfection, transfected cells were treated with 300 μM of KET for 12 hr.
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