Journal: Biochemical and biophysical research communications

Article Title: MicroRNA-133a-1 regulates inflammasome activation through uncoupling protein-2

doi: 10.1016/j.bbrc.2013.08.056

Figure Lengend Snippet: pCMV-UCP2-GFP or pCMV-GFP (vector) was transfected into differentiated THP1 cells seeded at 0.5 million cells/well in a six-well plate with coverslip. Untransfected cells acted as controls. (A) 48 hour post-transfection, cells were fixed in 4% paraformaldehyde, and the expression of GFP was examined under fluorescence microscope. (B) Expression of UCP2 in cell lysates transfected with UCP2-GFP or vector was analyzed by Western blot to confirm transfection. (C) After post-transfection with plasmids overexpressing UCP2 and controls, THP1 cells were stimulated with 5mM H2O2 and secretion of mature IL-β p17 and Caspase-1 p10 in the supernatants was compared by Western blot analysis.

Article Snippet: After 24 hours , the medium was replaced by serum-free RPMI-1640 and cells were transfected with 2.5μg/well DNA of pCMV-miR control (OriGene, Rockville, MD) and miR-133a-1 (Origene) plasmid using Lipofectamine® LTX plus Reagent (Invitrogen, CA, USA) as per manufacturer instructions.

Techniques: Plasmid Preparation, Transfection, Expressing, Fluorescence, Microscopy, Western Blot