For functional studies of RGS5, an siRNA-based silencing approach was used. Control siRNA (siGENOME Non-Targeting siRNA Pool #2, Thermo Scientific, Germany) and customized RGS5-targeting siRNA (sense: CCUGAAGUCUGAAUUCAGU, antisense: CCAUGAAUGUGGACUGGCA) was purchased from Sigma-Aldrich and used at a concentration of 100 nM. HUASMCs were transfected by utilizing the MATRAsi technique (IBA bioTAGnology, Göttingen, Germany) according to the manufacturer's instructions. Transfected cells were incubated for 48 h, and the siRNA-based knockdown efficiency was verified before further usage (Supplementary Fig S4).
Sigenome non targeting sirna pool 2
Manufactured by Thermo Fisher Scientific
Sourced in Germany
The SiGENOME Non-Targeting siRNA Pool #2 is a laboratory equipment product. It is a collection of small interfering RNAs (siRNAs) designed to not target any known gene in the human, mouse, or rat genome. This product is intended for use in RNA interference (RNAi) experiments as a control.
Automatically generated - may contain errors
Lab products found in correlation
Peretinoin, by Kowa (1 mentions)
Control sirna a, by Santa Cruz Biotechnology (1 mentions)
Sc 29352 sh, by Santa Cruz Biotechnology (1 mentions)
Lipofectamine 2000 transfection reagent, by Thermo Fisher Scientific (1 mentions)
X tremegene 9 dna transfection reagent, by Roche (1 mentions)
Lipofectamine rnaimax reagent, by Thermo Fisher Scientific (1 mentions)
A2058, by American Type Culture Collection (1 mentions)
Sk mel 28, by American Type Culture Collection (1 mentions)
Rnaimax, by Thermo Fisher Scientific (1 mentions)
Jetprime, by Polyplus Transfection (1 mentions)
5 protocols using sigenome non targeting sirna pool 2
1
RGS5 Silencing in HUASMCs
2
Modulating SPHK1 and Sp1 in Hepatocellular Carcinoma
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A mammalian expression plasmid of N-terminal myc-FLAG-tagged human SPHK1 (GenBank data base accession number NM_021972) and one encoding Sp1 under the control of cytomegalovirus promoter were purchased from OriGene (Rockville, MD). Three kinds of siRNAs targeting Sp1 (FlexiTube GeneSolution GS6667 SI#00150983, 02648065, and 02648072) were purchased from Qiagen (Hilden, Germany), as well as non-targeting siRNA (siGENOME Non-Targeting siRNA Pool #2, #D-001206-14-50) from Thermo Fisher Scientific (Waltham, MA). Peretinoin was kindly provided by Kowa Company (Aichi, Japan). Sphingosine kinase inhibitor 2 (SKI II, formally 4-[[4-(4-chlorophenyl)-2-thiazolyl]amino]-phenol) was purchased from Cayman Chemical Company (CAS No 312636-16-1; Ann Arbor, MI). These compounds were dissolved in DMSO. All final dilutions contained 0.5% DMSO. DEN was purchased from Sigma-Aldrich (St. Louis, MO).
3
Silencing HSC70 and HSP70 in Huh-7.5 Cells
4
siRNA Screening for Deubiquitinating Enzymes
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UACC257, M14, A2058, Malme-3M, SK-MEL-28, and MeWo cell lines were purchased from ATCC, and cultured in RPMI1640 supplemented with 2 mM l -glutamine, 10% fetal bovine serum, 100 units/mL of penicillin, and 100 μg/mL of streptomycin. The cells were maintained at 37 °C in a humidified atmosphere of 5% CO2.
For small interfering RNA (siRNA) screening, 12.5 nM Dharmacon siGENOME SMARTpool siRNA Library (Human Deubiquitinating Enzymes) and siGENOME Non-targeting siRNA Pool #2 (Thermo Fisher Scientific, Rockford, IL, USA) were reverse-transfected to UACC257 and M14 cells using Lipofectamine RNAiMAX reagent (Thermo Fisher Scientific). Other siRNAs were purchased from Thermo Fisher Scientific. For siRNA knockdown experiments, siRNA for PSMD14 (siPSMD14) (s19919 and s19920), siRNA for UBL5 (siUBL5) (s224521, s224522), siRNA for BAP1 (siBAP1) (s15821, s15822), siRNA for SMAD2 (s8397), siRNA for SMAD3 (s8402), or negative control #1 (siCNTL) was transfected at a final concentration of 12.5 nM into UACC257 or M14 cells using Lipofectamine RNAiMAX reagent. Transfected cells were subjected to CellTiter-Glo luminescent cell viability assay, Western blotting, and qRT-PCR after 96 h.
For small interfering RNA (siRNA) screening, 12.5 nM Dharmacon siGENOME SMARTpool siRNA Library (Human Deubiquitinating Enzymes) and siGENOME Non-targeting siRNA Pool #2 (Thermo Fisher Scientific, Rockford, IL, USA) were reverse-transfected to UACC257 and M14 cells using Lipofectamine RNAiMAX reagent (Thermo Fisher Scientific). Other siRNAs were purchased from Thermo Fisher Scientific. For siRNA knockdown experiments, siRNA for PSMD14 (siPSMD14) (s19919 and s19920), siRNA for UBL5 (siUBL5) (s224521, s224522), siRNA for BAP1 (siBAP1) (s15821, s15822), siRNA for SMAD2 (s8397), siRNA for SMAD3 (s8402), or negative control #1 (siCNTL) was transfected at a final concentration of 12.5 nM into UACC257 or M14 cells using Lipofectamine RNAiMAX reagent. Transfected cells were subjected to CellTiter-Glo luminescent cell viability assay, Western blotting, and qRT-PCR after 96 h.
5
Lentiviral and Retroviral Transduction for Stable Cell Lines
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