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. 2020 Sep 16;10(25):11535–11548. doi: 10.7150/thno.45261

Figure 5.

Figure 5

M2 macrophages promoted tumor cell proliferation and migration via the FAK/PI3K/AKT transduction signal pathway. (A) Cell viability of tumor cells detected using the XTT assay after treatment of M2 conditioned medium or control medium (*P < 0.05; ***P < 0.001; ****P < 0.0001). (B) Foci formation assay of tumor cells was conducted using M2 conditioned medium and control medium (***P < 0.001). The numbers of foci were calculated and are shown in the bar chart. (C) Representative images of IF showed the number of Ki67+ tumor cells after treatment of M2 conditioned medium and control medium (****P < 0.0001). The numbers of Ki67+ tumor cells were calculated and are shown in the bar chart. (D) The tumor volumes of excised tumors from mice injected with tumor cells stimulated by M2 conditioned medium and control medium. Linear graphs illustrate the growth rate of tumor after injection (n = 5 mice per group) (*P < 0.05; **P < 0.01). (E) Western blot results show the transduction signal pathway. The FAK-PI3K-AKT pathway was activated when tumor cells were stimulated by M2 conditioned medium. (F) Western blot results show the phosphorylation of AKT when tumor cells were treated with DMSO or LY294002 after being stimulated using M2 conditioned medium (RID: Relative Integrated Density). (H) Cell viability of tumor cells detected with the XTT assay after treatment of M2 conditioned medium combined with DMSO or LY294002 (*P < 0.05; **P < 0.01; ****P < 0.0001). (G) Foci formation assay of tumor cells was conducted using M2 conditioned medium with/without DMSO or LY294002 (***P < 0.001). The numbers of foci were calculated and are shown in the bar chart. (I) Representative images of IF showed the number of Ki67+ tumor cells after the treatment of the M2 conditioned medium with/without DMSO or LY294002 (**P < 0.01). The numbers of Ki67+ tumor cells were calculated and are shown in the bar chart. (J) Western blot results show the expression of CCL18 and Beta-actin in M0 macrophages and M2 macrophages. (K) Western blot results show the activation of the FAK-PI3K-AKT pathway when tumor cells were stimulated by PBS and 20 ng/µL and 100 ng/µL CCL18 recombinant.