Mirvana mirna mimic mir 1 positive control

On day 10, BMDCs were transfected with 50 nM of miR-369-3p mimic concentrations (Qiagen, Hilden, Germany) using TKO transfection reagent (Mirus Bio LLC, WI, USA), in accordance with the manufacturer’s instructions. Each transfection experiment was associated with mock control, where cells were transfected only with TKO transfection reagent (Mirus Bio LLC, WI, USA) without mimic, and with whole-cells control. The transfection efficiency was evaluated using a validated FAM-labeled miR-369-3p mimic (Thermo Fisher Scientific, MA, USA). In addition, the mirVana miRNA Mimic miR-1 Positive Control (Thermo Fisher Scientific, MA, USA) and the mirVana miRNA Mimic Negative Control #1 (Thermo Fisher Scientific, MA, USA) were used as positive and negative control, respectively. Bright‑field and fluorescence images were acquired using fluorescence Eclipse Ti2 microscope (Nikon Inc., Melville, NY). On day 12, BMDCs were stimulated whit 1 µg/ml LPS (Sigma-Aldrich, St. Louis, USA) and stored at 37 °C and 5% CO₂ for 24 or 48 h.

Transient transfection of miRNA mimic/inhibitor (50nM) (Ambion) was undertaken using Lipofectamine^® 2000 in presence of Opti-MEM Reduced Serum Medium (Thermo fisher Scientific). Concentrations of miRNA mimic/inhibitor and transfection reagents were optimized using BLOCK-iTTM Alexa Fluor^® Red Fluorescent Control (Ambion) as well as by mirVana^™ miRNA mimic miR-1 positive control and mirVana^™ miRNA Inhibitor, let-7c positive control. Expression of TWF1 in miR-1 mimic positive control transfected and HMGA2/MYC in let-7c inhibitor positive control transfected was detected by quantitative polymerase chain reaction (qPCR) to optimize transfection conditions.