According to the prediction results, wild-type (Wt) FOXO1 3ʹUTR or mutant (Mut) FOXO1 3ʹUTR sequence targeted by miR-1224-5p was synthesized and subjected to a luciferase reporter assay [39 (link)]. HEK-293 T cells (Zhong Qiao Xin Zhou Biotechnology, Shanghai, China) were incubated in DMEM supplemented with 10% FBS at 37°C. Cells were seeded in 12-well plates at a density of 5 × 104 and cultured in an incubator with 5% CO2 at 37°C. Before transfection, 293 T cells were incubated in a serum-free medium for 1 h. The cells were co-transfected with miR-1224-5p mimics or NC mimics and luciferase reporter plasmid containing wild-type (Wt) FOXO1 3ʹUTR or mutant (Mut) FOXO1 3ʹUTR using Lipofectamine 2000. After transfection for 48 h, 293 T cells were collected to detect firefly and Renilla luciferase activity using a Dual luciferase reporter gene assay kit (KeyGen Biotech, Nanjing, China).
Hek 293t cells
Manufactured by Shanghai Zhong Qiao Xin Zhou Biotechnology
Sourced in China
HEK-293T cells are a widely used human embryonic kidney cell line. They are a common tool in cell biology and biotechnology research, known for their high transfection efficiency and ability to produce recombinant proteins.
Automatically generated - may contain errors
Lab products found in correlation
Dual luciferase reporter gene assay kit, by Keygen Biotech (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Dmem f12 medium, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 2000 transfection reagent, by Thermo Fisher Scientific (1 mentions)
Hela cells, by Shanghai Zhong Qiao Xin Zhou Biotechnology (1 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
4 protocols using hek 293t cells
1
Investigating miR-1224-5p Regulation of FOXO1
2
Neuronal Cell Culture and CRISPR Manipulation
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P0 C57BL/6JGpt mice (provided by GemPharmatech) were sacrificed to obtain the hippocampi. The separated hippocampi were then digested with 0.25% trypsin and resuspended in DMEM/F12 medium (Gibco) containing 10% fetal bovine serum (FBS, Gibco). Cells were plated at a density of 106 cells/mL on poly‐D‐lysine‐precoated glass slides (Thermo Fisher Scientific). Within the first 4 hours of plating, the cells were cultured in 10% FBS‐DMEM/F12 medium, and then placed in Neurobasal‐based medium with 2% B27, 1% GlutaMAX, 37.5 mM NaCl, 0.3% W/V D‐glucose, and penicillin/streptomycin (commercial cell‐culture mediums and reagents are all from Gibco) for the rest of the culture period; half of the medium was exchanged every 2 days. The plasmid vector of CRISPR/Cas9 targeting the Nr4a1‐binding site of the TrkB promoter and the backbone Cas9 vector without the sgRNA array were transfected into primary cultured neurons with Lipofectamine 2000 transfection reagent (Invitrogen) 3 days after cell plating.
N2a, SH‐SY5Y neuroblastoma cells and HEK293T cells were purchased from Zhong Qiao Xin Zhou Biotechnology (China) and cultured in DMEM (Gibco) containing 10% FBS and 1% penicillin/streptomycin.
N2a, SH‐SY5Y neuroblastoma cells and HEK293T cells were purchased from Zhong Qiao Xin Zhou Biotechnology (China) and cultured in DMEM (Gibco) containing 10% FBS and 1% penicillin/streptomycin.
3
Cell Culture Protocol for Multiple Cell Lines
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Human embryonic kidney 293 T cells (HEK-293T cells), human cervical cells (HeLa cells), human breast cancer cells (MCF-7 cells) and mouse retinal endothelial cells (mREC cells), cultured in the Dulbecco’s modified Eagle’s medium with high glucose (H-DMEM), were purchased from Shanghai Zhong Qiao Xin Zhou Biotechnology Co., Ltd (China). All above-mentioned media were supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 1% relevant antibiotics (100 μg mL−1 streptomycin and 100 U mL−1 penicillin). All cell lines were cultured at 37 °C in a 5% CO2 incubator with the humidified atmosphere.
4
Regulation of KLF7 by miR-9a-5p
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HEK-293T cells (Zhong Qiao Xin Zhou Biotechnology Co., Ltd., Shanghai, China) were grown in 12-well plate to 70% confluence and serum starved for 1 h. Then they were co-transfected with
miR-9a-5p mimic or control mimic and wild type 3’UTR of Klf7 or 3’UTR mutation of Klf7 using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) according to
the manufacturer’s instructions. After transfection, luciferase activities were measured by Dual Luciferase kit (KeyGen, Nanjing, China).
For following experiment, the FLS cells were transfected with miR-9a-5p inhibitor, inhibitor control, miR-9a-5p mimic or mimic control to evaluate the role of miR-9a-5p in regulating KLF7
expression. Then FLS cells were transfected with negative control inhibitor, miR-9a-5p inhibitor, miR-9a-5p inhibitor and NC siRNA, or miR-9a-5p inhibitor and KLF7 siRNA followed by TNF-α
stimulation for 24 h to verify whether the biological function of KLF7 was mediated by miR-9a-5p. The miR-9a-5p mimics, miR-9a-5p inhibitors and their negative control were purchased from
General Biological System (Anhui) Co., Ltd. (Anhui, China).
miR-9a-5p mimic or control mimic and wild type 3’UTR of Klf7 or 3’UTR mutation of Klf7 using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) according to
the manufacturer’s instructions. After transfection, luciferase activities were measured by Dual Luciferase kit (KeyGen, Nanjing, China).
For following experiment, the FLS cells were transfected with miR-9a-5p inhibitor, inhibitor control, miR-9a-5p mimic or mimic control to evaluate the role of miR-9a-5p in regulating KLF7
expression. Then FLS cells were transfected with negative control inhibitor, miR-9a-5p inhibitor, miR-9a-5p inhibitor and NC siRNA, or miR-9a-5p inhibitor and KLF7 siRNA followed by TNF-α
stimulation for 24 h to verify whether the biological function of KLF7 was mediated by miR-9a-5p. The miR-9a-5p mimics, miR-9a-5p inhibitors and their negative control were purchased from
General Biological System (Anhui) Co., Ltd. (Anhui, China).
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