Journal: Scientific Reports

Article Title: Vesicle miR-195 derived from Endothelial Cells Inhibits Expression of Serotonin Transporter in Vessel Smooth Muscle Cells

doi: 10.1038/srep43546

Figure Lengend Snippet: ( a ) Left: western blot analysis of exosome protein. Full-length blots are presented in . Right: Electron microscopy image of isolated extracellular vesicles of ECs (30–100 nm). ( b ) Schematic of the degradation assay used in this study. ( c ) EC-CM was incubated with the indicated reagents cultured SMCs. The protein of SMCs was extracted 48 hours later and the levels of 5-HTT was measured by WB. Full-length blots are presented in . ( d )Semi-quantification results of 5-HTT protein level after cultured with different treated EC-CM. **P < 0.01 compared with control (n = 3).

Article Snippet: Human umbilical vein endothelial cells (HUVEC) and human umbilical vein smooth muscle cells (HUVSMCs) were freshly isolated from human umbilical cord vein as previously described and cultured with ECs growth medium (Promo Cell) and DMEM/F12 supplemented with 10% FCS, 1% sodium pyruvate, 1% glutamine and 1% penicillin and streptomycin respectively.

Techniques: Western Blot, Electron Microscopy, Isolation, Degradation Assay, Incubation, Cell Culture

Journal: Scientific Reports

Article Title: Vesicle miR-195 derived from Endothelial Cells Inhibits Expression of Serotonin Transporter in Vessel Smooth Muscle Cells

doi: 10.1038/srep43546

Figure Lengend Snippet: ( a ) 5-HTT expression was inhibited after miR-15, miR-195, miR16, miR-322 mimics transfection in SMCs. The micro-RNA mimics were transfected into SMCs and the protein was extracted after 48 hours. Full-length blots are presented in . **P < 0.01 compared with control (n = 3). ( b ) The miRNA level of miR-15 and miR-195 were measured by real-time PCR in normal and injured carotid artery. **P < 0.01 compared with shame (n = 3). ( c ) SMCs was cultured by EC-CM 24 hours when RNA was isolated. The miRNA level was detected by real time PCR. **P < 0.01 compared with control (n = 4). ( d ) The miRNAs expression level of extracellular vesicles from ECs was measured by real-time PCR (n = 3). ( e ) Extracellular vesicles of ECs were incubated with the indicated reagents before isolating RNA and measuring the levels of the indicated miRNAs by real-time PCR. **P < 0.01 compared with control (n = 4). ( f ) SMCs were treated with EC-CM from FAM-linked miRNA transfected ECs. FAM-linked miRNA in ECs and SMCs was detected by fluorescence microscopy. ( g ) ECs were transfected with miR-195 inhibitors and the EC-CM were used to culture SMCs. The protein of SMCs was extracted 48 hours later and the levels of 5-HTT was measured by WB. Full-length blots are presented in .

Article Snippet: Human umbilical vein endothelial cells (HUVEC) and human umbilical vein smooth muscle cells (HUVSMCs) were freshly isolated from human umbilical cord vein as previously described and cultured with ECs growth medium (Promo Cell) and DMEM/F12 supplemented with 10% FCS, 1% sodium pyruvate, 1% glutamine and 1% penicillin and streptomycin respectively.

Techniques: Expressing, Transfection, Real-time Polymerase Chain Reaction, Cell Culture, Isolation, Incubation, Fluorescence, Microscopy