Mcpip1 sirna

Cell culture medium and reagents, oligofectamine, lipofectamine 2000, random hexamers, turbo DNA-free kit, miRNA assay kit, miRNA reverse transcription kit, synthetic mature and pre-mature miRNAs, anti-miR-200 oligonucleotides, MCPiP1 siRNA, PCR TaqMan probes, Go TaqH hot start DNA polymerase and Moloney Murine Leukemia Virus Reverse Transcriptase (MMLV) were provided by Life Technologies. Gemcitabine (GEM), 5-Azacytidine (5-AZA) and anti-β-actine antibody were from Sigma-Aldrich. Enhanced chemiluminescent substrate was from Millipore. Protease inhibitor cocktail was provided by Roche Life Science. Kapa Fast Sybr qPCR Mix was from Clinisciences. RNAzol-RT, RanQ69L-GTP and anti-Dicer1 antibody were from Euromedex. GoTaq G2 DNA polymerase, Go Script reverse transcriptase and RNAsin RNase inhibitor were from Promega. PCR, qPCR and RT specific primers were obtained from Eurogentec. PolyA polymerase tailing kit, anti-MCPiP1, anti-Histone H1 and anti-calnexin antibodies were from Tebu-Bio. Anti-XPO₅ antibody was from Cell Signalling. Protein A/G plus agarose was provided by Santa Cruz. pDestmycDICER1 expression vector was a gift from Thomas Tuschi (Addgene plasmid # 19873).

Primary culture of mouse cortical neurons was prepared as previously reported (Hilgenberg and Smith, 2007 (link)) with some modifications. Briefly, the mouse brain cortex was digested in 0.25% trypsin at 37°C for 10 min. The cell suspension was centrifuged at 1,000 rpm for 5 min. The pellet was resuspended in Neurobasal-A/B-27 medium (Thermo Fisher Scientific) with 0.25 mmol/L Glutamax-1, 40 U/mL penicillin, 40 μg/ml streptomycin, and 10% dialyzed horse serum. Cells were plated in poly-L-lysine-coated 24-well plates at 6 × 10⁵ cells per well, and cultured at 37°C in a humidified chamber containing 5% CO₂.
Transfections of MCPIP1 siRNA (4,390,771, Life Technologies) or control siRNA (4,390,843, Life Technologies) were performed as previously reported (Liang et al., 2008 (link)) using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer’s instructions. 24 h later after 25 nM of the siRNA transfection, the TMP (10 μM) was added to the culture medium for 12 h, and the cells were subjected to OGD protocol as previously described (Gong et al., 2014 (link)). Briefly, the cells were cultured with glucose-free medium with 95% N₂, 5% CO₂ at 37°C for 120 min. The culture medium was then replaced with normal culture medium and incubated under normal conditions for 24 h. Cells without OGD were used as controls.