Macrophages promote inflammation, stemness, and Snail1 expression, and downregulates USP4 in lung cancer cells. (A) LLC lung cancer cells were co-cultured with or without bone marrow-derived macrophages (BMDMs) in 0.4 μm transwell plates as illustrated. (B) Expression levels of stemness-associated genes were analyzed by RT-qPCR. (C) Cell surface stemness marker CD117 analyzed by flow cytometry. Left panels show a set of the histograms. (D) Expression levels of inflammatory cytokines analyzed by RT-qPCR. (E,F) Expression of Snail1 and USP4 at the mRNA level (E) and protein level (F) analyzed with RT-qPCR and immunoblotting, respectively. (G) Inflammatory cytokines upregulate Snail1 and downregulate USP4 in lung cancer cells. D121 and H1299 were treated with TNF-α (20 ng/mL) or IL-1β (20 ng/mL) for 48 h. Data presented as mean ± SD of three independent experiments. * P < 0.05; ** P < 0.01.