Sigenome risc free control sirna

SiRNAs were diluted in Lipofectamine RNAiMAX transfection reagent (Invitrogen) and incubated for 15 min at room temperature before being added to cells in fresh culture medium. Invalidation of atg5 was performed using 25 nM of a pool of four siRNAs (M-004374-04-0005, Dharmacon (Lafayette, LA, USA), Target sequences: 5′-GGAAUAUCCUGCAGAAGAA-3′—5′-CAUCUGAGCUACCCGGAUA-3′—5′-GACAAGAAGACAUUAGUGA-3′—5′-CAAUUGGUUUGCUAUUUGA-3′) or two different siRNAs from the on-target plus set of four siRNAs (LU-004374-00-0005, Dharmacon, Target sequences: #7: 5′-GGCAUUAUCCAAUUGGUUU-3′—#10: 5′-ACAAAGAUGUGCUUCGAGA-3′). A non-targeting siRNA pool (siGENOME RISC-Free Control siRNA, Dharmacon) was used as a control. Transfections were stopped after 24 h by renewing the culture medium.
3-MA (Sigma-Aldrich, St. Louis, MO, USA; M9281) and Bafilomycin A1 from Streptomyces griseus (B 1793), respectively, diluted in water and DMSO, were directly added to the culture medium at 1 or 5 mM for 3 MA and 5 nM for Bafilomycin and let to react for 48 h.

ON-TARGETplus SMARTpool siRNA targeted to DNMT1 (Dharmacon, Austin, TX) and siGENOME RISC-Free Control siRNA (Dharmacon, Austin, TX) were used. RCC4-VHL cells grown on 10 cm tissue culture plates were transfected with control or DNMT1 siRNA at a final concentration of 200 nM using Oligofectamine as per manufactures instructions.

siGENOME RISC-Free Control siRNA (Dharmacon) and Silencer Select Pre-designed siRNAs against mouse ASPP2 (#4390771, siRNA s102092, Ambion) were resuspended in nuclease-free sterile water and used at 20 μM. For zygotes, 3 to 4-week-old CD-1 females (Charles River UK) were injected intraperitoneally with 5 IU of PMSG (Intervet) and 48 h later with 5 IU of hCG (Intervet), and were paired with C57Bl/6 J male mice (in house). Zygotes were retrieved from oviductal ampullae at 20 h post-hCG. Cumulus-enclosed zygotes were denuded by exposure to 1 mg/mL hyaluronidase (Sigma) in modified mHTF (Life Global) containing 3 mg/ml BSA for 3–6 min and cultured in LGGG-020 (life Global) containing 3 mg/ml BSA in the presence of 5% CO₂ at 37 °C. Microinjection of zygotes commenced 2 h after release from cumulus mass. Zygotes with normal morphology were microinjected into the cytoplasm in 30 µl drops of modified HTF media containing 4 mg/ml BSA using a PMM-150FU Piezo impact drive (Primetech) using homemade glass capillaries with ∼5–10 pl of siRNA. Zygotes were returned to LGGG-020 containing 3 mg/ml BSA in the presence of 5% CO₂ at 37 °C until analysis.

HCT116 cells were reverse-transfected with Lipofectamine RNAiMax (Invitrogen) according to manufacturer’s instructions using 5 nM small interfering RNA targeting OGT (siGENOME human OGT siRNA D-019111-01, Dharmacon), EZH2 (EZH2 siGENOME SMART Pool M-004218-03-0005, Dharmacon) or a scrambled control sequence (siCTRL; siGENOME RISC free control siRNA, Dharmacon) as previously described [19 (link)]. Seventy-two hours later, cells were harvested for RNA/protein extraction.

GloResponse NF-κB-RE-luc2P HEK293 cells were seeded at 10,000 cells/well in 96-well plates. After 24 hr, cells were transfected with siRNA at a final concentration of 25 nmol/l with Dharmafect reagent #1. After 72 hr, cells were stimulated with TNF and analyzed by NF-κB luciferase reporter assay. For signaling pathway analysis, HEK293 cells at a density of 100,000 cells/well in 24-well plates were transfected as before and stimulated with TNF and lysed in the presence of protease and phosphatase inhibitor cocktail for immunoblot. Western blot gel electrophoresis, transfer, and detection was performed in parallel with identical film exposure duration. The following siRNA sequences were used: UBE2L3 sense 5′-CCGCAAAUGUGGGAUGAAA-3′, anti-sense 5′-UUUCAUCCCACAUUUGCGG-3′; HOIL-1 sense 5′-GCUCAGAUGCACACCGUCA-3′ and anti-sense 5′-UGACGGUGUGCAUCUGAGC-3′; HOIP sense 5′-GGCGUGGUGUCAAGUUUAA-3′ and anti-sense 5′-UUAAACUUGACACCACGCC-3′; and Sharpin sense 5′-CCUGGAAACUUGACGGAGA-3′ and anti-sense 5′-UCUCCGUCAAGUUUCCAGG-3′. siGENOME RISC-Free Control siRNA (Dharmacon) was used as a control.

HEK293T cells were reverse-transfected with Lipofectamine RNAiMax (Invitrogen) according to manufacturer’s instructions using 10 nM small interfering RNA targeting HIC1 (Human, Dharmacon), or a scrambled control sequence (si Ctrl; siGENOME RISC free control siRNA, Dharmacon). 48 hours after siRNA transfection, cells were treated for RNA extraction [25 (link)].