Fig. 1: Polarity and immune response gene signatures of in vitro polarised macrophages and TAMs from s.c. melanoma.

A–C Macrophages differentiated from the bone marrow of C57BL/6 mice were polarised in vitro toward M1 with 20 ng/ml IFN-γ plus 0.1 μg/ml LPS (M_IFN-γ+LPS) and toward M2 with 20 ng/ml IL-4 (M_IL-4) for 36 h, or left unpolarised. Total RNA was isolated and subjected to a customised RT-qPCR array of mouse immune response and macrophage polarity genes. Newly induced genes are displayed as average relative expressions + standard deviation (SD), n = 3 (A). Relative expression of regulated genes in M_IFN-γ+LPS (B) and in M_IL-4 (C) is displayed as fold changes over the relative gene expression of unpolarised macrophages. Means of changes of 5-fold or greater + SD are shown, n = 3. D A representative image of CD11b immunostaining (red) of s.c. B16F10 melanoma from C57BL/6 mouse (nuclei are stained blue with DAPI), and a representative dot plot of fluorescence activated cell sorting of F4/80⁺CD11b⁺ TAMs from the tumour. E, F Total RNA was isolated from pooled TAMs of s.c. tumours (n = 3) and subjected to a customised RT-qPCR array of mouse immune response and macrophage polarity genes. Newly induced genes are displayed as average relative expressions + SD, n = 3 (E). Relative expression of regulated genes is shown as fold changes over the relative gene expression of naïve infiltrating (interstitial) lung macrophages. Means of changes of 5-fold or greater + SD are presented, n = 3 (F). G Venn diagram showing the overlap between upregulated genes of M_IFN-γ+LPS, M_IL-4 and TAMs.