Mir21 mimic

miR21-silenced MLO-Y4 osteocytic cells were generated using short hairpin (sh)RNA Lentiviral Particles (Sigma-Aldrich Chemical Co., St. Louis, MO, USA), as previously reported,[5 ,44 ] and cultured as previously described. MLO-Y4 osteocytic cells silenced or not for Cx43 were plated at the density of 2 × 10⁴ cells/cm⁻² on 48-well plates coated with type I rat tail collagen and cultured overnight. Cells were transiently transfected using Lipofectamine RNAiMAX reagent (Invitrogen) containing miRIDIAN negative control inhibitor, miR21 inhibitor, negative control mimic, or miR21 mimic (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA) at a final concentration of 0.1 nM in medium without serum and penicillin/ streptomycin for 6 h. Next, 2x concentrated medium was added to each well and then cultured overnight. Medium was changed to a regular growing medium, and cells were then cultured for an additional 24 h before isolating mRNA. miR21 levels were decreased by 60% in MLO-Y4 scramble cells and by 91% in MLO-Y4 Cx43 shRNA cells treated with miR21 inhibitor, as measured by qPCR. miR21 levels were increased by 44% in MLO-Y4 scramble cells and 234% in MLO-Y4 Cx43 shRNA cells treated with the miR21 mimic.