Retinal endothelial cells, prepared from bovine eyes, from 4–7th passage were incubated in normal (5mM) or high (20mM) D-glucose for 4 days, and parallel osmotic controls included cells incubated in 20mM mannitol or 20mM L-glucose 9 (link), 20 (link) To investigate the effect of inhibition of Tet in demethylation of MMP-9 promoter, a batch of cells were incubated with a cell-permeable inhibitor (2S)-Octyl-α-hydroxyglutarate (2-HG, 500μM; Cayman, Ann Arbor, MI) 29 (link). The cells received fresh media, including 2-HG, every 24 hours. Inhibition of Dnmts/Tets was further confirmed by transfecting the cells with their specific siRNAs (Dnmt1-siRNA, Santa Cruz Biotechnology, Santa Cruz, CA, and Tet2-siRNA, Integrative DNA Technology, Coralville, IA) using Lipofectamine® RNAiMAX transfection reagent (Thermo Fisher Scientific, IL), as routinely performed in our laboratory 9 (link), 20 (link). Parallel incubations with non-targeting scrambled RNA were used as transfection controls. After transfection, the cells were rinsed and incubated in either 5mM or 20mM D-glucose media for 4 days. The efficiency of transfection was determined by quantifying their protein (western blot) and gene (SYBR Green-based quantitative real-time PCR, qPCR) expressions.
Dnmt1 sirna
Manufactured by Santa Cruz Biotechnology
Sourced in United States
DNMT1 siRNA is a small interfering RNA (siRNA) designed to target the DNA methyltransferase 1 (DNMT1) gene. DNMT1 is responsible for maintaining DNA methylation patterns during cell division. The DNMT1 siRNA can be used to knockdown the expression of the DNMT1 gene in cell-based experiments.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine rnaimax transfection reagent, by Thermo Fisher Scientific (4 mentions)
2s octyl α hydroxyglutarate 2 hg, by Cayman Chemical (2 mentions)
Block it alexa fluor red fluorescent oligo, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Lipostem, by Thermo Fisher Scientific (1 mentions)
Trichostatin a tsa, by Merck Group (1 mentions)
Anti hdac1, by Santa Cruz Biotechnology (1 mentions)
Anti pericentrin, by Abcam (1 mentions)
Anti e2f1, by Abcam (1 mentions)
Goat anti rabbit igg hrp antibody, by Santa Cruz Biotechnology (1 mentions)
Anti dnmt1, by Santa Cruz Biotechnology (1 mentions)
Mouse monoclonal anti β actin, by Santa Cruz Biotechnology (1 mentions)
Anti cdk2, by Abcam (1 mentions)
Cdna synthesis kit, by Merck Group (1 mentions)
Anti γ tubulin, by Abcam (1 mentions)
Control sirna, by Santa Cruz Biotechnology (1 mentions)
Anti n cadherin, by Abcam (1 mentions)
Anti bcl 2, by Abcam (1 mentions)
Anti c myc, by Abcam (1 mentions)
Fetal bovine serum (fbs), by HiMedia (1 mentions)
5 protocols using dnmt1 sirna
1
Retinal Endothelial Cell Epigenetics
2
Retinal Endothelial Cell Epigenetics
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Retinal endothelial cells, prepared from bovine eyes, from 4–7th passage were incubated in normal (5mM) or high (20mM) D-glucose for 4 days, and parallel osmotic controls included cells incubated in 20mM mannitol or 20mM L-glucose 9 (link), 20 (link) To investigate the effect of inhibition of Tet in demethylation of MMP-9 promoter, a batch of cells were incubated with a cell-permeable inhibitor (2S)-Octyl-α-hydroxyglutarate (2-HG, 500μM; Cayman, Ann Arbor, MI) 29 (link). The cells received fresh media, including 2-HG, every 24 hours. Inhibition of Dnmts/Tets was further confirmed by transfecting the cells with their specific siRNAs (Dnmt1-siRNA, Santa Cruz Biotechnology, Santa Cruz, CA, and Tet2-siRNA, Integrative DNA Technology, Coralville, IA) using Lipofectamine® RNAiMAX transfection reagent (Thermo Fisher Scientific, IL), as routinely performed in our laboratory 9 (link), 20 (link). Parallel incubations with non-targeting scrambled RNA were used as transfection controls. After transfection, the cells were rinsed and incubated in either 5mM or 20mM D-glucose media for 4 days. The efficiency of transfection was determined by quantifying their protein (western blot) and gene (SYBR Green-based quantitative real-time PCR, qPCR) expressions.
3
iPSC-derived Neuron Transfection Protocol
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iPSC-derived neurons were transfected with LipoStem (Thermofisher Scientific) on day 13 or 14 of differentiation according to the manufacturer’s protocol. Half of the culture medium was removed and stored before transfection. Transfection of cells was conducted using 15 nM control siRNA (BLOCK-iT Alexa Fluor red fluorescent oligo, 14750100; Invitrogen (Carlsbad, CA, USA)) or 30 nM DNMT1 siRNA (sc-35204; Santa Cruz Biotechnology). Then, 24 h after transfection, the medium was changed to previously collected conditioned medium, and cells were transferred to a live-imaging chamber (ALA Scientific Instruments, Farmingdale, NY, USA) to perform live-cell calcium imaging. To roughly estimate the transfection efficiency, cotransfection of cells was conducted using 15 nM control siRNA and 30 nM DNMT1 siRNA as above, and transfected cells were manually counted based on the presence of red fluorescence, yielding a transfection efficiency of 39.1 ± 2.3% 24 h after transfection.
4
Molecular Techniques for Epigenetic Analysis
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All the chemicals used in this study were molecular biology grade as well as endotoxin free and used without further purification. Culture media, fetal bovine serum (FBS) and trypsin were purchased from Himedia and antibiotic from GIBCO. Trichostatin A (TSA), reagents for RNA isolation, cDNA synthesis Kit, Syber green for qRT-PCR, gene specific primer pairs were obtained from Sigma-Aldrich, St. Louis, MO, USA. Anti-DNMT1, anti-HDAC1, mouse monoclonal anti-β-actin and Goat anti-rabbit IgG-HRP antibodies, DNMT1 siRNA and scrambled (control) siRNA were purchased from Santa-Cruz Biotechnology. Anti-PARP (cleaved), anti-HDAC2, anti-E-cadherin, anti-N-cadherin, anti-vimentin, anti-α-tubulin, anti-γ-tubulin, anti-pericentrin, anti-TUBGCP2, anti-c-Myc, anti-Ras, anti-Cdk2 and anti-E2F1, anti-Bcl2 and anti-Bax antibodies were obtained from abcam.
5
siRNA-mediated DNMT1 Knockdown in HEK293T Cells
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Human embryonic kidney 293T (HEK) cells were cultured in high-glucose DMEM (Invitrogen) supplemented with 10% FBS at 37 °C, 95% humidity and 5% CO2. Prior to any treatment, cells were seeded into 6-well plates at a density of 8 × 104 cells per well.
The siRNA transfection was performed 24 h after seeding, using Lipofectamine 2000 (Thermo Fisher Scientific) according to the manufacturer’s protocol for reverse lipofection and 15 nM control siRNA (BLOCK-iT Alexa Fluor red fluorescent oligo, 14750100; Invitrogen) or 30 nM DNMT1 siRNA (sc-35204; Santa Cruz Biotechnology) for 5 h in Opti-MEM I reduced serum medium (Thermo Fisher Scientific). Subsequently, cells were again cultured in the previously described medium for 24 h at 37 °C, 95% humidity and 5% CO2.
The siRNA transfection was performed 24 h after seeding, using Lipofectamine 2000 (Thermo Fisher Scientific) according to the manufacturer’s protocol for reverse lipofection and 15 nM control siRNA (BLOCK-iT Alexa Fluor red fluorescent oligo, 14750100; Invitrogen) or 30 nM DNMT1 siRNA (sc-35204; Santa Cruz Biotechnology) for 5 h in Opti-MEM I reduced serum medium (Thermo Fisher Scientific). Subsequently, cells were again cultured in the previously described medium for 24 h at 37 °C, 95% humidity and 5% CO2.
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