Fig. 6. TNF-α-driven CCL4 expression in human monocytic cells involves activation of the NF-κB/AP1 activity. THP-1-XBlue cells i.e. THP-1 cells stably expressing a SEAP (secreted embryonic alkaline phosphatase) reporter inducible by NF-κB/AP-1 promoter activity, were incubated with TNF-α (10ng/ml) or LPS (10ng/ml; positive control) for 24 hrs while mock-treated cells served as negative control. Cell culture media were assayed for SEAP reporter activity (OD detected at 650nm wavelength) as a measure of the degree of NF-κB/AP-1 activation. CCL4 gene expression in THP-1-XBlue cell lysates and CCL4 secreted protein expression in culture supernatants were assessed by real-time RT-PCR and ELISA, respectively. The data show (A) significantly enhanced activation of NF-κB/AP-1activity in THP-1-XBlue cells treated with TNF-α compared to mock-treated control (P=0.0198). Also, the data show concomitantly upregulated CCL4 (B) gene (P=0.0008) and (C) secreted protein (P=0.001) expression in TNF-α stimulated THP-1-XBlue cells compared to mock-treated controls.