Figure 4

Concentration-dependent effects of DOX on RAW 264.7 macrophages. Cells were exposed to increasing concentrations of DOX (0.005, 0.01, 0.05, 0.1, or 0.5 µM) for 24 h and cell cytotoxicity was measured using MTT assay (A). In (B, C and D), cells were exposed to DOX (0.1 µM) or LPS (10 ng/mL) plus IFN-γ (10 U/mL). Nitrite production was determined using Griess method (B). mRNA levels of iNOS, TNF-α, IL-6 and TLR4 were measured using qPCR and were normalized to GAPDH (C). Protein levels of TNF-α and IL-6 were quantified using ELISA (D). Data are expressed as a percentage of control (at 100%) ± S.E. (n = 8) for cell viability, or as mean ± S.E. (n = 3) for nitrite, qPCR and ELISA. Comparisons are made with ANOVA followed by Student–Newman–Keuls (SNK) post-hoc test; *, P < 0.05, compared with control; #, P < 0.05, compared with LPS/IFN-γ group.