OPN gene expression and protein secretion from monocytes cocultured with HS-27a. (A) CD14⁺ cells were cocultured in contact with HS-27a (contact coculture) or separated from HS-27a by a Transwell insert (transwell coculture). Controls included CD14 alone in a Transwell insert, CD14 alone, and HS-27a alone. Cells were cultured for 3 days. Both adherent and nonadherent monocytes were harvested and combined, except in the contact culture where only the nonadherent cells were harvested. Total RNA was harvested, and OPN gene expression was determined by Syber Green PCR assay and normalized to G3PDH gene expression. (B) Conditioned media were also harvested after 3 days, and concentrations of OPN protein were determined by ELISA. (C) CD14⁺ cells were cultured in multiwell culture plates in the absence or presence of conditioned media from HS-27a at different concentrations for 3 days. Adherent and nonadherent CD14⁺ cells were combined, and total RNA was isolated. OPN gene expression was determined as described above. The data represent the average ± SD for at least 2 samples.