Research Paper Advance Articles pp 24394—24423

E3 ligase FBXW7 restricts M2-like tumor-associated macrophage polarization by targeting c-Myc

Figure 5. FBXW7 knockout promotes macrophage expressing pro-tumoral factors. (A) BMDMs from FBXW7f/f and Lysm+FBXW7f/f mice were stimulated with conditioned medium, and the mRNA expression of MMP9, IL-10, TGFβ, and VEGFα was examined by qRT-PCR. (B) The protein expression of MMP9 and VEGFα in BMDMs incubated with conditioned medium were detected by immunoblotting. (C) The protein levels of IL10 and TGFβ in the supernatant of BMDMs which co-cultured with LLCs for indicated time were measured by ELISA kit. (D) LLCs were cultured in serum-free RPMI-1640, supernatant from IL-4-induced wild-type or FBXW7-knockout macrophages. The proliferation of LLCs in three groups was measured by MTT assay. (E) Schematic representation of the co-injection experimental approach. IL4-induced BMDMs derived from FBXW7f/f and Lysm+FBXW7f/f mice were mixed with LLCs at a ratio of 1:1 and injected subcutaneously into healthy wild-type C57BL/6 mice. (F, G) The volume (F) and weight (G) of tumors in co-injection mice. (H) The appearance of tumors in two groups inoculated with a mixture of M2 macrophages and LLCs. Data are shown as the mean ± SD and are representative of three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 (two-way ANOVA (A, C, D, F) and Student’s t test (G)).