Fig. 4. TNF-α-induced CCL4 expression is downmodulated by genetic suppression of SAPK/JNK signaling. THP-1 monocytic cells were transfected with SAPK/JNK siRNA while cells transfected with scrambled siRNA served as mock-treated control as described in materials and methods. Later cells were stimulated with TNF-α and incubated at 37°C for 24 hrs. Cells and supernatants were collected and CCL4 mRNA in cell lysates and protein expression in culture supernatants were determined using real-time RT-PCR and ELISA, respectively. (A) The data show that SAPK/JNK siRNA transfection in human monocytic cells resulted in significant target gene suppression compared to mock-treated control (P=0.0024). TNF-α stimulation of THP-1 cells transfected with SAPK/JNK siRNA resulted in significant suppression of CCL4 (B) gene (P=0.0108) and (C) protein (P=0.0151) expression compared to mock-treated controls. (D) SAPK/JNK kinase assay confirmed the c-Jun phosphorylation in TNF-α-treated cells compared to mock-treated controls while palmitate treatment served as a positive control.