Fig. 1. TNF-α upregulates CCL4 expression in human monocytic cells and derived macrophages. THP-1 cells were treated with TNF-α (10 ng/ml) or LPS (10ng/ml; positive control) for 24 hrs. Cells and culture media were collected. Total RNA was isolated and CCL4 mRNA was quantified by real-time RT-PCR. Relative mRNA expression was expressed as fold change over average gene expression in mock-treated controls. Secreted CCL4 protein was measured in cell supernatants using ELISA. To use THP-1 derived macrophages, THP-1 monocytic cells were differentiated into macrophages with PMA (50 ng/ml) for 72 hours and then treated with TNF-α and LPS for 24 hrs as described earlier. Cells and culture media were collected. CCL4 mRNA expression in cell lysates and secreted CCL4 protein in cell supernatants were measured as described. The data show that CCL4 (A) gene (P=0.0026) and (B) protein (P=0.0025) expression was significantly higher in THP-1 monocytic cells treated with TNF-α compared to controls. Similarly, CCL4 (C) gene (P=0.0065) and (D) protein (P=0.0022) expression was significantly upregulation in THP-1-derived TNF-α-treated macrophages compared to controls.