Fig. 4.

Lipopolysaccharide (LPS)-induced monocyte chemoattractant protein 1 (MCP-1) production is osteopontin (OPN)-dependent in U937. (A) Messenger RNA (mRNA) and protein (D) expression of MCP-1 from U937 cells in response to LPS (0.01, 0.1, and 1 µg/mL) were detected by real-time polymerase chain reaction (RT-PCR) at 8 h and ELISA at 24 h. The U937 cells were stimulated with LPS (1 µg/mL) after pretreatment with OPN siRNA. Total RNA was extracted and subjected to reverse transcription followed by RT-PCR to detect MCP-1 mRNA at 8 h (B). MCP-1 in cell-free supernatants after 24 h stimulated by LPS was assayed using ELISA (E). The U937 cells were stimulated with OPN (5, 50, and 500 ng/mL) for 8 h to detect MCP-1 mRNA (C) or 24 h to detect its secretion of MCP-1 in supernatants (F). Each data point represents mean ± standard error of the mean of 3 experiments. * and ** stand for P values less than 0.05 and 0.01, as compared to the control; + and ++ stand for P values less than 0.05 and 0.01, as compared to LPS (1 µg/mL), respectively.