Construction of plasmids and site directed mutagenesis For plasmid DNA and miRNA co transfection, primary chondrocytes were transfected using the Human Chon drocyte Nucleofector kit following the manufacturers instructions. The miR 146a expression plasmid was created as previously described. Briefly, the precursor sequence for miR 146a was amplified through PCR using genomic DNA necessary as the template, and the PCR Inhibitors,Modulators,Libraries products were cloned into the pSuper vector Luciferase reporter assay All plasmids for transfection were prepared using the QIAGEN plasmid purification kit. HEK293T cells were transiently transfected using Lipofectamine 2000 according to the manufacturers instructions, and pRL SV40 vector was used as a control for transfection efficiency.
Twenty four hours after transfec tion, cells were lysed, and Firefly and Renilla luciferase activities were measured using the Dual Luciferase Reporter Assay System according to the man ufacturers protocol. C5. 18 cells were co transfected with Inhibitors,Modulators,Libraries miR 146a mimics and p3TP lux using DharmaFECT Inhibitors,Modulators,Libraries Duo transfection reagent. The p3TP lux plasmid was a kind gift from Dr Regis J. OKeefe. Twelve hours after trans fection, the cells were serum starved for 12 hours fol lowed by 4 hours treatment with or without TGF b1. Cell lysates were extracted and luciferase activities were measured using the Dual Luci ferase Reporter Assay System. Each experi ment was repeated at least three times. RNA and quantitative real time PCR Total RNA, including miRNA, was extracted using the miRNeasy Mini Kit according to the manu facturers instructions.
Then 1 ug total RNA was reverse transcribed with a specific stem loop primer for miRNA and with a random primer for mRNA, respec tively. After RT reaction, real time PCR was performed by an ABI 7900HT system using Inhibitors,Modulators,Libraries SYBR Premix Ex Taq. b actin and small nuclear RNA U6 were Inhibitors,Modulators,Libraries used as internal controls for cDNA and miRNA, respectively. Primer sequences used for real time PCR are presented in Table 1. Western blotting Whole cell lysates were prepared with ice cold lysis buf fer supplemented with protease inhibitors. Proteins were size fractionated by SDS PAGE and transferred to a PVDF membrane. Membranes were hybridized with antibodies against Smad4, VEGF, extracellular signal regulated kinase 1 2, phospho ERK1 2 and GAPDH. Densitometric analysis of immunoblots was per formed using the ImageJ software provided by the National Institutes of Health.
Smad4 knockdown by siRNA RNA interference was performed using siGENOME SMARTpool siRNA targeting rat Smad4. http://www.selleckchem.com/products/baricitinib-ly3009104.html Transfection for primary chondrocytes was car ried out using Lipofectamine RNAiMAX reagent according to the manufacturers protocol. TUNEL assay Chondrocytes were fixed for 20 minutes at room tem perature with 4% paraformaldehyde in PBS 48 hours post transfection, and apoptosis was assessed using the In Situ Cell Death Detection Kit Fluorescein according to the manufacturers instructions.