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. 2020 Jul 29;9(8):1799. doi: 10.3390/cells9081799

Figure 4.

Figure 4

MIP-1α co-induction by palmitate and TNF-α involves the IRF3 pathway. To investigate whether the MIP-1α co-induction by palmitate and TNF-α required MyD88 adaptor protein for TLR4-downstream signaling, we used MyD88-null THP1-XBlue-defMyD cells and treated cell cultures at 37 °C with palmitate (200 μM) and/or TNF-α (10 ng/mL) for 24 h. Cell pellets were collected for total RNA extraction for measuring MIP-1α gene expression and cells supernatants were collected for measuring MIP-1α secreted protein as described in the Materials and Methods section. The representative data (mean ± SEM) from three independent determinations with similar results show that MIP-1α co-induction by palmitate and TNF-α was sustained in MyD88-defective cells which showed elevated expression of (A) MIP-1α transcripts (P < 0.0001) and (B) MIP-1α secreted protein (P < 0.0001) in response to co-stimulation with palmitate and TNF-α as compared to those treated with either TNF-α or palmitate. On the contrary, siRNA-mediated ablation of IRF3 resulted in the diminution of (C) MIP-1α mRNA (P = 0.006) and (D) MIP-1α secreted protein (P = 0.001) compared to controls transfected with scrambled siRNA. Thus, MIP-1α co-induction in THP-1 cells by palmitate and TNF-α involves the IRF3-mediated signaling. ** P < 0.01 and **** P < 0.0001.