To assess the protective effects of xenon, a cohort of A549 cultures were treated with TNF-α (2 ng/ml; Sigma-Altrich, United Kingdom) or H 2 O 2 (100 μM) for 24 h (Sigma-Altrich). Another cohort of cultures were treated with human HIF-1α small interfering RNA (siRNA) or scrambled siRNA (Qiagen, United Kingdom) for 6 h before gas exposure or an inhibitor of PI3K, LY294002 (Cell Signaling, United Kingdom) at 20 μM or dimethyl sulfoxide (DMSO) vehicle for 16 h before gas exposure or an inhibitor of mTOR rapamycin (Tocris Bioscience, United Kingdom) 100 nM or DMSO vehicle after gas exposure. In vitro HIF-1α siRNA transfections were carried out using lipofectamine (Invitrogen). siRNA targeting human HIF-1α (Qiagen; sense strand; 5'-GAAGAACUAUGAACAUAAATT-3' and antisense strand: 5'-UUUAUGUUCAUAGUUCUUCCT-3') were administered to A549 cells in a dose of 20 nM. scrambled siRNA served as a negative control. Cells were incubated with siRNA for 6 h at 37°C in humidified air containing 5% CO 2 , after which they were then incubated with experimental medium followed by xenon gas treatment.
Scrambled sirna
Manufactured by Qiagen
Sourced in Germany, United States, United Kingdom
Scrambled siRNA is a laboratory tool used to create a negative control for RNA interference (RNAi) experiments. It is a synthetic double-stranded RNA molecule designed to have no known complementary targets in the target organism's genome, serving as a reference to distinguish specific gene silencing effects from non-specific or off-target effects.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine rnaimax, by Thermo Fisher Scientific (10 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (9 mentions)
Lipofectamine rnaimax reagent, by Thermo Fisher Scientific (5 mentions)
Hiperfect transfection reagent, by Qiagen (4 mentions)
Lipofectamine, by Thermo Fisher Scientific (3 mentions)
Opti mem, by Thermo Fisher Scientific (3 mentions)
Amaxa cell line nucleo factor kit r, by Lonza (2 mentions)
Miro2, by Qiagen (2 mentions)
Incucyte live cell imager, by Sartorius (2 mentions)
Mvi sirna, by Qiagen (2 mentions)
Flexitube sirna premix, by Qiagen (2 mentions)
Glucocorticoid receptor sirna, by Santa Cruz Biotechnology (2 mentions)
Diaph1 8 flexitube sirna, by Qiagen (2 mentions)
Opti mem reduced serum medium, by Thermo Fisher Scientific (2 mentions)
Mission sirna transfection reagent, by Merck Group (2 mentions)
Ripa buffer, by Merck Group (2 mentions)
Turbofect, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Lipofectamine transfection reagent, by Thermo Fisher Scientific (1 mentions)
61 protocols using scrambled sirna
1
Xenon's Protective Effects on A549 Cells
2
Electroporation and siRNA Transfection for B Lymphoma
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The Amaxa Cell Line Nucleo- factor Kit R (L-013 program; Lonza) was used to electroporate 5 × 106 IIA1.6 B Lymphoma cells for different transfections. For Centrin-GFP plasmid transfection we used 2 µg of DNA, and cells were cultured for 16 h before functional analysis. In the case of transfection with siRNA we used Silencer® Select (Ambion/Thermofisher) against Nesprin-1(Syne1): s234287 (#1), s234288 (#2); siRNA Sun1: s94911 (#1), s94912 cells (#2), these were incubated for 48 h before analysis. As a control, we used a scrambled siRNA (Qiagen) at 10nM.
3
Silencing Pcaf in Vascular SMCs
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Vascular SMCs were transfected using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions with control short-interfering RNA (scrambled siRNA; Qiagen) or a combination of 4 siRNAs directed towards Pcaf (Qiagen) for 4 hours. Subsequently vSMCs were stimulated with LPS (1 ng/ml) for 24 hours and supernatants were collected and analyzed by ELISA. To confirm Pcaf knockdown, qPCR was used to analyze Pcaf expression. RNA was isolated using RNeasy minikits (Qiagen) and qPCR was performed on ABI7500 Fast system using Taqman gene expression assays for Hprt1 and Pcaf.
4
HepG2 Cultures: Histone, VEGF, and NLRP3/AIM2 Regulation
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Some cohort cultures of HepG2 cultures were treated with histone H3 recombinant protein (20 μg/mL; Sigma‐Aldrich, Poole, UK), some cohort cultures were treated with VEGF recombinant protein (5 ng⁄mL; Thermo Fisher Scientific, Paisley, UK) or PBS vehicle for 24 hours, and some cohort cultures were treated with human nucleotide‐binding domain, leucine‐rich repeat containing protein 3 (NLRP3) siRNA (SI03060323; Qiagen, Crawley, West Sussex, UK), AIM2 siRNA (SI04261432; Qiagen), VEGF siRNA (sc‐29520; Santa Cruz Biotechnology, Dallas, TX), VEGFR1 siRNA (sc‐29319; Santa Cruz), VEGFR2 siRNA (sc‐29318; Santa Cruz), or scrambled siRNA (Qiagen) 6 hours before experiments. siRNA was dissolved in siRNA suspension buffer and administered to HepG2 cells at a dose of 20 nmol/L; scrambled siRNA served as a negative control. The transfection was carried out through highly efficient Lipofectamine Transfection Reagent (Thermo Fisher Scientific UK).21 Cells were incubated with siRNA for 6 hours before histone treatment. Some cell cohort received caspase‐1 inhibitor Ac‐YVAD‐CHO (20 μmol/L; Santa Cruz).
5
Primary Human Retinal Endothelial Cell Culture
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Primary human RECs were purchased from Cell System Corporation (CSC, Kirkland, WA). The cells were grown in normal glucose medium (Cell Systems) with the addition of microvascular growth supplements (Invitrogen), 10 μg/ml gentamycin, and 0.25 μg/ml amphotericin B. Once confluent, some cells were transferred to high (25 mM) glucose medium (Cell Systems) for 3 days. Only primary cells within passage 6 were used. Cells were quiesced by incubating in a high or normal glucose medium without growth factors for 24 h and used to perform the experiments unless otherwise indicated.
Some cells were treated with an Epac1 agonist (8-CPT-2’-O-Me-cAMP, 10 μM, 24 h), as we have done previously [14 (link)]. Additional cells in high glucose were transfected with TNFAIP3 siRNA (Qiagen, Germantown, MD) or scrambled siRNA (Qiagen).
Some cells were treated with an Epac1 agonist (8-CPT-2’-O-Me-cAMP, 10 μM, 24 h), as we have done previously [14 (link)]. Additional cells in high glucose were transfected with TNFAIP3 siRNA (Qiagen, Germantown, MD) or scrambled siRNA (Qiagen).
6
Knockdown of MRTF-A in VSMCs
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VSMCs were transfected with short interfering RNA (siRNA) directed against MRTF-A as previously described.21 (link) Scrambled siRNA (Qiagen) was used as a control. Transfection with siRNA targeting zyxin was done as previously described.4 (link) For each well of a 6-well plate, 3 μg of siRNA was diluted in Opti-MEM I (Invitrogen) using 3 μL of MATra-si reagent (IBA) giving a final volume of 200 μL. After incubating the mixture for 20 minutes at ambient temperature, the solution was added onto the cells, which had been pre-incubated in 2 mL Opti-MEM I prior to the transfection. Cells were then incubated on a magnetic plate (IBA) at 37°C and 5% CO2. After 20 minutes, cells were washed and normal cell medium was added to the cells followed by incubation for 48 hours for knockdown of MRTF-A.
7
Downregulation of AdipoR1/AdipoR2 in BV2 Cells
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AdipoR1 or AdipoR2 interfering RNA (siRNA) or scrambled siRNA (Qiagen) were transfected into BV2 cells using RNAiMax Lipofectamine (Invitrogen) according to the manufacturer's recommendations. After various experimental tests, 48 h post-transfection time was selected as the optimal duration to allow maximal decrease of targeted protein expression. Therefore, in all experiments, cells were used 48 h after siRNA transfection.
8
Silencing ATG5 in DLD-1 cells
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Small interfering RNAs (siRNAs) targeting ATG5 (NM_004849) and scrambled siRNA were purchased from QIAGEN (Hilden, Germany). The siRNA target sequences were as follows: ATG5#1 siRNA 5′-AACCTTTGGCCTAAGAAGAAA-3′ and ATG5#2 5′-CTAGGAGATCTCCTCAAAGAA-3′. DLD-1 cells at 60–70% confluence were transfected with a 10 nM final concentration of siRNA using the HiPerFect Transfection Reagent (QIAGEN). Twenty-four hours after transfection, protein and total RNA was extracted.
9
ELOVL4 Overexpression and Knockdown in SHSY-5Y Cells
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SHSY-5Y neuronal cells were cultured in a 12-well plate in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum, 1% Glutamax, 0.5% glucose, 100 IU/ml penicillin and 100 μg/ml streptomycin at 37 °C in humidified air containing 5% CO2. Knockdown of ELOVL4 was performed using siRNA from Qiagen (Product no. 1027416) and Lipofectamine 2000 as per the manufacturer’s protocol. Scrambled siRNA (Qiagen, Australia) was used as a negative control. Overexpression of ELOVL4 was performed using an ELOVL4 expressing plasmid from Origene (Cat no. RC206248) and Lipofectamine 2000 as per the manufacturer’s protocol. Empty vector, pcDNA (Invitrogen, Australia), was used as a negative control.
10
Cenp-F and Miro2 Knockdown Assay
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Oligonucleotides for siRNA synthesized against the target sequence of Cenp-F (oligo #1: 5′-CAGGAAAGACTAGCCCATATA-3′, oligo #2: 5′-CAGAATCTTAGTAGTCAAGTA-3′, oligo #3 (ineffective): 5′-CTGGTGATGGATTAACATATA-3′, oligo #4: 5′-ACCGAGAGAAATTGACTTCTA-3′), Miro2 (oligo #1 5′-AAGGCAGAGCTTTGGGCCAAA-3′, oligo #2 5′-GAGGTTGGGTTCCTGA-TTAAA-3′) and scrambled siRNA 5′-TTCTCCGAACGTGTCACGT-3′ were purchased from Qiagen. siRNA transfection were performed using Lipofectamine RNAiMAX (Life Technologies), according to the manufacturer, at a final concentration of 30 nM.
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