The role of macrophage MyD88 and TNFα in MCP-1 mRNA expression by LLC cells. (A) One thousand LLC cells were seeded into 12-well plates. After overnight incubation at 37°C, three different numbers of PC from WT, MyD88−/− or TNF−/− mice were added to the wells. To neutralize TNFα, 2 or 10 μg of anti-mouse TNFα IgG (R&D Systems) was added with 1 × 105 PC from WT mouse. After incubation at 37°C for 5 days, total RNA was isolated and the expression of MCP-1 mRNA was examined by Northern blotting (10 μg per lane). (B) The experiment presented in A was repeated with 1 × 105 PC from WT, MyD88−/− or TNF−/− mice and the MCP-1 concentration in the culture supernatants was measured by ELISA. The results are shown as the mean ± SEM. *p < 0.0001, n = 4. (C) The expression of TNF mRNA in 4T1 tumors or LLC tumors was examined by Nanostring gene profiling. The results are shown as the mean ± SEM.
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