Gene expression and secretion of OPN. (A) HS-27a cells were cultured alone or cocultured with CD14⁺ cells at a HS-27a/CD14 ratio of 2:1 for 3 days. Adherent and nonadherent cells were harvested separately from the coculture. Total RNA was isolated and reverse-transcribed into cDNA. OPN gene expression was determined by a Syber Green real-time PCR assay and normalized to G3PDH gene expression. (B) CD14⁺ cells (0.2 × 10⁶ cells) were cultured in the presence (○) or absence (▪) of HS-27a (0.4 × 10⁶ cells) for 3 days. Nonadherent cells were harvested daily, and OPN gene expression was determined as described for panelA. (C) CD14⁺ cells from 13 different healthy donors were cultured alone (CD14 cells alone) or cocultured with HS-27a (CD14 cells in coculture) for 3 days. The nonadherent monocytes were harvested, and OPN gene expression was determined by a Syber Green assay as above. (D) CD14⁺ cells (0-0.2 × 10⁶ cells) were cultured alone (CD14 alone) or cocultured with 0.4 × 10⁶ cells of HS-27a (coculture) for 3 days. ELISA was used to determine the concentration of OPN in conditioned media. Each symbol represents the average ± SD for 3 samples.