The TLR4 inhibitor TAK-242 and the antioxidant NAC attenuate heme-induced M1 polarization of macrophages. (A,D) M0, (B,E) M1, and (C,F) M2 BMDMs were left untreated (NT) or exposed to 5 μM heme-BSA (+Heme; heme-BSA 1:1 ratio) with or without (A-C) 400 nM TAK-242 and (D-F) 2 mM NAC for 12 hours. M1 and M2 markers are shown in the left and right column of each panel, respectively. The expression of MHCII, CD14, and CD206 was analyzed by flow cytometry and expressed in RFU as fold change to nontreated (NT) cells. mRNA levels of TNFα, Arginase-1, and IL-10 were analyzed by qRT-PCR and expressed in RQ, as fold change to untreated (NT) cells. Results shown are representative of 3 independent experiments. Values represent mean ± SEM. *P < .05; **P < .01; ***P < .001; ****P < .0001.