Fig. 6

S1P-ALOX15 signaling mediated TAMs. A Computer-imaging analysis for the 12/15-LOX positive cells and F4/80 positive cells detected by IHC staining in the tumor tissues from xenograft model of EL4-WSHFD mice and EL4-CD mice. B Representative images of IHC in serial sections from the same paraffin tissue block to detect 12/15-LOX positive cells and F4/80 positive cells from the xenograft model of EL4-WSHFD mice and EL4-CD mice. C Dual immunofluorescent staining using the antibodies of anti-F4/80 and anti-12/15-LOX to confirm the co-localization of 12/15-LOX positive cells and F4/80 positive cells in isolated macrophages from EL4-WSHFD mice in comparison with EL4 cells. D The protein levels of phosphorylated SPHK1 and SPHK1 by Western blot in lymphoma cells (HH and SU-DHL-4) co-cultured with THP-1 cells and the S1P levels by the enzyme-linked immunosorbent assay (ELISA) in the supernatant of co-culture medium. E The mRNA levels of S1P receptor (1–5) by q-PCR in the THP-1 monocytes, M0 phenotype induced by PMA, M1 phenotype induced by LPS, and M1 phenotype induced by S1P. F The mRNA levels of ALOX15 in the THP-1 monocytes treated with LPS, S1P, co-culture medium, and inhibitors of S1P receptors. G The mRNA levels of arginase-1, TGFβ, and PD-L1 in the THP-1 monocytes treated with LPS, S1P, inhibitors of S1P receptors, and inhibitors of ALOX15. H The tumor volume changes after xenograft and tumor mass weight measured at sacrifice in 12/15-LOX^−/−-EL4-WSHFD mice and WT-EL4-CD mice. I: Flow Cytometry analysis to detect the subpopulations of F4/80⁺CD11b⁺ cells and F4/80⁺CD206⁺ cells for potential TAMs in the collected the PMM from the peritoneal injection model of 12/15-LOX^−/−-EL4-WSHFD mice and WT-EL4-CD mice. CM: co-culture medium; PMM: peritoneal monocytes/macrophages; HPF: high-power field. Scale bar = 100 μm in IHC staining. Scale bar = 50 μm in dual immunofluorescent staining. *, p < 0.05; **, p < 0.01; ***, p < 0.001