The HNSCC cell lines (SAS and Sa3) used in this study were obtained from the RIKEN BioResource Center (Tsukuba, Ibaraki, Japan). The miRNA precursors, negative control miRNA, and siRNAs were obtained from Applied Biosystems (Waltham, MA, USA). The procedures used for the transient transfection of miRNAs, siRNAs, and plasmid vectors were described in our previous studies [20 (link),21 (link),57 (link),58 (link)]. miRNAs at 10 nM and siRNAs at 5 nM were transfected into HNSCC cell lines using RNAiMAX reagent (Invitrogen, Waltham, MA, USA). The reagents used in this study are listed in Supplemental Table S1 ; here, “mock”: transfection reagent only, and “control”: negative control miRNA precursor that have no function transfected.
Sirnas
Manufactured by Thermo Fisher Scientific
Sourced in United States, China, United Kingdom, Germany
SiRNAs are a class of double-stranded RNA molecules that are used to silence gene expression. They work by binding to and degrading specific mRNA molecules, preventing them from being translated into proteins. SiRNAs are a versatile tool for researchers in the life sciences, as they can be used to study gene function and to develop potential therapeutic interventions.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (59 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (58 mentions)
Lipofectamine rnaimax reagent, by Thermo Fisher Scientific (13 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (13 mentions)
Sirnas, by GenePharma (12 mentions)
Control sirna, by Thermo Fisher Scientific (10 mentions)
Lipofectamine rnaimax transfection reagent, by Thermo Fisher Scientific (10 mentions)
Sirnas, by RiboBio (10 mentions)
Opti mem, by Thermo Fisher Scientific (9 mentions)
Rnaimax, by Thermo Fisher Scientific (8 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (5 mentions)
Sirnas, by Merck Group (5 mentions)
Neon transfection system, by Thermo Fisher Scientific (5 mentions)
Scrambled sirna, by Thermo Fisher Scientific (4 mentions)
Sirnas, by Santa Cruz Biotechnology (4 mentions)
Dharmafect 1, by Thermo Fisher Scientific (4 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (4 mentions)
Rnaimax transfection reagent, by Thermo Fisher Scientific (4 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (3 mentions)
Si nc, by Thermo Fisher Scientific (3 mentions)
258 protocols using sirnas
1
Transient Transfection of miRNAs and siRNAs in HNSCC Cells
2
Stable Cell Lines for miRNA-124 Overexpression
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Stable H522M3/pRTR-miR-124 and H1975M3/pRTR-miR-124 cells were generated by transfection of the episomal expression vector pRTR using Fugene6 (Roche). The cells were then selected in the presence of 3.0 μg/mL puromycin (Sigma) and validated by flow cytometry for GFP-positive cells 72 h after addition of doxycycline (Sigma). pre-miRNAs (P-miRNAs), anti-miRNA, siRNAs and respective negative controls (Applied Biosystems, Foster City, CA, USA) were transfected into cells using Lipofectamine 2000 Reagent (Invitrogen) according to the manufacturer's protocol.
3
Monocyte and Alveolar Macrophage Signaling
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Monocytes and AMs were cultured overnight with inhibitors added 1 hour prior to stimulation. The cells were then stimulated with LPS (Sigma), or oxidants as detailed in the figures. The expression of selected proteins was knocked down by siRNAs (Applied Biosystems) using lipofectamine 2000 (Invitrogen). The siRNAs against p38α MAPK, MSK-1, MSK-2, IKK-1 and IKK-2 were introduced to the cells as per the manufacturer’s instructions and the effective knockdown of protein levels was observed 72h after transfection as assessed by Western blotting for the respective target protein levels.
4
Epithelial-Mesenchymal Transition Regulation
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The HCC cell lines J7, HepG2, and SK-Hep1 were cultured in DMEM medium containing 10% fetal bovine serum at 37°C in a 5% CO2 atmosphere. Polyclonal antibodies against Hsp90, HIF-1α, E-cadherin, N-cadherin, vimentin, occludin, twist, snail and β-actin were purchased from Genetex (Irvine, CA) and Cell Signaling Technology (Beverly, MA). Secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). All siRNAs were purchased from Applied Biosystems (Foster City, CA). pCDNA3.1-CPS1-IT1, a CMV-based expression and neomycin-selective plasmid containing lncRNA-CPS1-IT1, was constructed by GenScript Co. (Piscataway, NJ).
5
Efficient Knockdown of GRK2 Protein
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RASM (2 × 10 6 ) cells were transfected with 10 nM negative-control (non-targeting sequence) or GRK2 (5′-GCAGGUACCUCCAGAUCUCtt-3′) small interfering (si)RNAs (Applied Biosystems, UK), as previously validated by our group [2, 28] . siRNAs were introduced using the Lonza nucleofection system (Lonza, Cologne, Germany) according to the manufacturer's instructions. Transfected cells were grown for 48 h to allow for efficient knockdown of the target protein expression before experiments were performed. Specific protein depletion was quantified using standard western blotting techniques, exactly as described previously [2, 3] .
6
Hypoxia Response in HUVECs
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HUVECs were transfected using Lipofectamine RNAiMAX (Thermo Scientific) according to the manufacturer’s protocol. All siRNAs (Ambion, Austin, TX, USA) were used at a final concentration of 40 nM: XBP1 (ID s14915), ERN1 (ID s200432), and Negative Control No. 1 (#4390843). After 24 h, the transfected cells were put into a hypoxia chamber for 6 h, whereas the control cells remained in an incubator with normoxic conditions.
7
Targeting key cellular pathways
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Small interfering RNA (siRNAs) targeting IL-6 (S7312) and Bcl-XL (120717) were purchased from Ambion (Austin, TX, USA). siRNAs targeting SOD2 (sc-41655), β-catenin (sc-44275), and STAT3 (sc-29209) as well as lentiviruses expressing small hairpin RNAs (shRNAs) targeting SOD2 (sc-41655-V), Bcl-XL (sc-43630-V), and SULF2 (sc-63088-V) were obtained from Santa Cruz Biotechnology.
8
Silencing ELAVL1 Gene in Cell Cultures
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siRNAs were from Ambion. For the ELAVL1 (HUR) gene we used siELAV1-1 (invitrogen #s4609) sense 5′-UGAACUACGUGACCGCGAAtt-3′, antisense 5′-UUCGCGGUCACGUAGUUCAca-3′; siELAV1-2 (invitrogen #s4610) sense 5′-GCGUUUAUCCGGUUUGACAtt-3′, antisense 5′-UGUCAAACCGGAUAAACGCaa-3′. For silencing experiments, 2,000,000 cells were transfected with 400 nM of siRNA using GenePulser XcellTM (Bio-Rad, Madrid, Spain) with 300 V and 1000 μF and incubated for 48 h.
9
Synthesis and Delivery of TAT-Mediated siRNA
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Synthesis of TAT was performed as described in a previous study (16 (link)). In brief, 50 nM siRNAs (Ambion; Thermo Fisher Scientific, Inc.) were dissolved in RNase-free ddH2O and then mixed with 10 µM TAT at a TAT:siRNA molar ratio of 20:1 and incubated for 30 min at 37°C. The siRNA sequences are shown in Table II . For in vivo use, treatment of the tumors was initiated at 2 weeks after tumorsphere implantation, as previously described (17 (link)). In brief, dissociated cells from tumorspheres were injected into the left and right sides of the mouse (as described above), and palpable tumors were formed 2 weeks later. At this time point, the tumor site on one side of the mouse was injected with TAT-mediated si-hWAPL (100 µM TAT/siRNA) and that on the other side was injected with control [scrambled (Scr) siRNA]. The tumor size was measured each week.
10
Knockdown of Oncogenes in Ovarian Cancer
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Knockdown of PRKCZ, IGF1R, and ITGB3 expression in ovarian cancer cell lines was achieved by transfection of siRNAs (Ambion). siRNAs targeting of these genes was performed with Dharmafect-4 transfection reagent (Dharmacon). In brief, cells were seeded in 12-well or 6-well plates at densities of 1 x 105 or 2 x 105 cells/well, respectively. Cells were then treated with siRNA transfection mixtures following the manufacturer’s protocol. Scrambled siRNA (Ambion) was used as a control. Additional controls included mock-treated cells that received transfection reagent without siRNA, as well as untreated cells that received only fresh media. Cells were harvested after 48 or 72 hours for RNA and protein extraction, respectively.
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