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. 2019 Sep 19;20(18):4658. doi: 10.3390/ijms20184658

Figure 2.

Figure 2

CCL4 co-induction by TNF-α/palmitate is TLR4 dependent. To investigate the involvement of TLR4 in the cooperative induction of CCL4 by TNF-α and palmitate, TLR4 signaling activity was blunted in THP-1 cells by TLR4 siRNA, the TLR4 neutralizing antibody or the TLR4 chemical inhibitor OxPAPC as described in the materials and methods section. The data (mean ± SEM) show that the cooperative effect of TNF-α/palmitate co-treatment was predominantly lost following siRNA-mediated ablation of TLR4 in THP-1 cells, resulting in significantly reduced expression of (A) CCL4 mRNA (p = 0.019) and (B) protein (p = 0.026) compared to control (scramble siRNA). Similarly, the cooperative induction of CCL4 by TNF-α/palmitate was abrogated after the TLR4 receptor was intercepted by treatment with an anti-TLR4 neutralizing antibody, displaying the diminished expression of (C) CCL4 mRNA (p = 0.002) and (D) CCL4 protein (p = 0.003) compared to the control (isotype-matched antibody). In addition, THP-1 cells treated with the the TLR4 inhibitor OxPAPC before TNF-α/palmitate co-stimulation also exhibited reduced expression of (E) CCL4 mRNA (p = 0.004) and (F) secreted CCL4 protein (p = 0.006) compared to the control (mock treatment). * Significant (p < 0.03), ** Very significant (p < 0.007).