Panx3 hemichannel releases intracellular ATP and promotes differentiation. (A) Imaging of intracellular ATP levels in pEF1- or pEF1/Panx3-transfected C2C12 cells. Cells were incubated with the caged luciferin, and then exposed to a flash of UV light for photolysis to convert active luciferin. Fluorescence excitation images (red) caused by luciferin–ATP interactions at 5 s and 15 s after a UV flash were shown. Higher red fluorescence images were observed in control pEF1-transfected cells compared with Panx3-overexpressing cells, which indicates that Panx3 overexpression reduced intracellular ATP levels. (A, b and c) Measurement of ATP release. The transfected C2C12 cells or primary calvarial cells were treated with or without KGlu for 2 min, and then ATP released into the media was measured. (B) Inhibition of ATP release. The transfected cells were incubated with Panx3 antibody or Panx3 peptide for 30 min, and ATP release was measured. The Panx3 antibody (1.5 µg/ml) inhibited ATP release in pEF1/Panx3-transfected cells (a). This inhibition was blocked by various concentrations of the Panx3 peptide. The Panx3 peptide also inhibited the ATP release (a). Control sh- or shPanx3-transfected cells were cultured with BMP2 for 2 d, and ATP release was measured. ATP release was reduced in shPanx3-transfected cells compared with control sh-transfected cells (b). (C) Inhibition of osterix (a) and ALP (b) expression by the anti-Panx3 antibody. *, P < 0.05; **, P < 0.01. Error bars represent the mean ± SD; n = 3.