Rat articular chondrocytes were isolated from femoral heads, femoral condyles, and tibial plateaus of rats as described previously.40 (link) Cultured chondrocytes were maintained as a monolayer in DMEM/F12 (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) with 10% fetal bovine serum (Gibco), 100 U/ml penicillin G, 100 μg/ml streptomycin, and 2.5 μg/ml amphotericin B under a humidified atmosphere of 5% CO2 in air at 37 °C. Recombinant TNF-α (10 ng/ml; 300-01A; PeproTech, Rocky Hill, NJ, USA) was used to stimulate chondrocytes in complete medium. miRNA mimics or inhibitor (GenePharma, Shanghai, China) and siRNAs (GenePharma) were transfected with Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions at a concentration of 40 nM. Sequences of miRNA mimics and inhibitor are shown in Supplementary Table 1 .
Mirna mimics or inhibitor
Manufactured by GenePharma
Sourced in China
MiRNA mimics or inhibitors are synthetic RNA molecules designed to either mimic or inhibit the function of endogenous microRNAs (miRNAs) in biological systems. These molecules are used as research tools to study the roles of specific miRNAs in cellular processes and gene expression regulation.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Sirnas, by GenePharma (2 mentions)
Recombinant tnf α, by Thermo Fisher Scientific (1 mentions)
Dmem f12, by Thermo Fisher Scientific (1 mentions)
Luciferase activity assay kit, by Beyotime (1 mentions)
Pmirglo control vector, by Promega (1 mentions)
Microplate reader, by Molecular Devices (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 3000 reagent, by Thermo Fisher Scientific (1 mentions)
Lentivirus constructs, by Genechem (1 mentions)
5 protocols using mirna mimics or inhibitor
1
Rat Articular Chondrocyte Isolation and Culture
2
3'-UTR LIP mRNA Luciferase Assay
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The 3′-UTR LIP mRNA wild-type and mutated sequences (GenBank accession No. MT176433) were cloned into the pmirGLO control vector (Promega, Madison, WI, USA) to obtain the pmirGLO-lip-3′-UTR-WT/MUT plasmids, respectively. The same sequences were cloned into the pEGFP-C1 vector to obtain the pEGFP-lip-3′-UTR-WT/MUT plasmids, respectively.
HEK293 cells were seeded on 48-well plates overnight before transfection and then transfected with 25 ng of dual-luciferase reporter WT/MUT vector and 50 nM of miRNA mimics or inhibitor (GenePharma, Suzhou, CHN). Dual-luciferase reporter assays were performed 24 h after transfection using the luciferase activity assay kit, following the manufacturer’s instructions (Beyotime, Beijing, CHN). Firefly luciferase activity was normalized to Renilla luciferase activity. Relative light unit (RLU) data were measured using a microplate reader (Molecular Devices, San Jose, CA, USA) in luminescence detection mode.
HEK293 cells were seeded on 48-well plates overnight before transfection and then transfected with 25 ng of dual-luciferase reporter WT/MUT vector and 50 nM of miRNA mimics or inhibitor (GenePharma, Suzhou, CHN). Dual-luciferase reporter assays were performed 24 h after transfection using the luciferase activity assay kit, following the manufacturer’s instructions (Beyotime, Beijing, CHN). Firefly luciferase activity was normalized to Renilla luciferase activity. Relative light unit (RLU) data were measured using a microplate reader (Molecular Devices, San Jose, CA, USA) in luminescence detection mode.
3
Plasmid and miRNA Transfection in HEK-293T Cells
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Plasmids and 100 mM miRNA mimics or inhibitors (Genepharma, Shanghai, China) were transfected into HEK-293T cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol. Cells were collected 48 h later for luciferase assays.
4
Cell Culture Methodology for Colon Cancer
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All cell lines used in this study were purchased from the Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China) and verified by short-tandem repeat (STR) profiling. All cells were cultured in the appropriate medium (RPMI-1640 for NCM460, SW480, HT29, HCT15 and HCT116; L-15 for SW620; DMEM for Caco2; and F-12 K for LOVO) supplemented with 10% FBS (Gibco, Carlsbad, CA, USA) in a humidified atmosphere with 5% CO2 at 37 °C. Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) was used for transient transfection of small RNA oligos and plasmids. For miRNA overexpression or knockdown, miRNA mimics or inhibitors (GenePharma, Shanghai, China) were used, respectively. For protein overexpression or knockdown, gene-specific overexpression vectors (FulenGen, Guangzhou, China) or siRNAs (GenePharma) were used, respectively. The siRNA sequences are listed in Additional file 2 : Table S2.
5
Lentivirus-Mediated Modulation of FTO
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The lentivirus constructs of FTO overexpression or knockdown were purchased from Genechem (Shanghai, China). Briefly, T24, 5637 and UM-UC-3 were plated in six-well dishes and infected with FTO-overexpression lentivirus, negative control, FTO knockdown lentivirus and scramble control at 40% confluence. The viral titre was determined according to the manufacturer’s instructions. Seventy-two hours after transfection, puromycin (2 μg/mL) was used to select stable transductions.
The miRNA mimics or inhibitors were purchased from GenePharma (Shanghai, China) and were transfected into the cells using Lipofectamine 3000 reagent (Invitrogen, USA).
All lentivirus constructs, mimics, negative control, and inhibitor sequences are listed in Supplementary Table2 .
The miRNA mimics or inhibitors were purchased from GenePharma (Shanghai, China) and were transfected into the cells using Lipofectamine 3000 reagent (Invitrogen, USA).
All lentivirus constructs, mimics, negative control, and inhibitor sequences are listed in Supplementary Table
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