Red siglo oligos

MiScript miRNA mimics (miR-24, -30b, -101, 142-3p, -652-3p, -652-5p, and -1275) and inhibitors were purchased from Qiagen (Germantown, MD, USA). For control, all stars negative mimics (Qiagen) were used. For PKCα knockdown, gene specific and control siRNA were purchased from Sigma (St. Louis, MO, USA). Transient transfections were performed using Lipofectamine 2000 (Life Technologies) according to manufacturer's instructions. Mφ were transfected with mimics or inhibitors at a final concentration of 50 nM while DC, monocytes and PBMCs were transfected at a final concentration of 100 nM. Red siGLO oligos (ThermoScientific, Waltham, MA, USA) were used to determine transfection efficiency.

miR-30b, miR-142–3p and control miRNA mimic were purchased from Qiagen (Gaithersburg, MD, USA). Transient miRNA overexpression was performed by transfecting miRNA mimics using Lipofectamine 2000 (Invitrogen-Life Technologies Corporation, Carlsbad, CA, USA) according to the manufacturer’s instructions. Day 7-differentiated MΦ or DC were transfected at a final concentration of 50 nM. Red siGLO oligos (ThermoFisher Scientific, Grand Island, NY, USA) were used to determine transfection efficiency.

Transient transfections were performed using Lipofectaine 2000 reagent (Life Technologies, San Diego, CA) according to manufacturer’s instructions. Red siGLO oligos (ThermoScientific, Waltham, MA) were used to determine transfection efficiency. After 36 hours, cells were harvested for RNA isolation. Cells were transfected with viral miRNA mimics at increasing concentrations of 10, 20 and 50 nM (to determine dose effects) for 36 hours. We noticed robust (~90%) transfection at 20 nM, therefore, we used the same concentration for most of the experiments unless specified. Controls consisted of mock transfected and control mimic (Qiagen) transfected cultures.