Figure 3

M1 type macrophages induced by 4Mu ameliorate hepatocellular carcinoma aggressiveness. (a) Hepa129 cells were incubated for duplicate with RPMI (Hepa129 + RPMI), CM from isolated pMϕ (Hepa129 + pMϕ-derived CM), and CM from isolated pMϕ in vitro treated with 4Mu (Hepa129 + 4Mu-treated pMϕ-derived CM) for 48 h. Pre-conditioned 1 × 10⁶ Hepa 129 cells were injected s.c. in C3Hj mice (n = 4/group), and tumor volume was measured. (b) Hepa129 cells were cultured with RPMI, pMϕ-derived CM and 4Mu-treated pMϕ-derived CM, and the expression levels of TLR4, CD47 and Sox2 were analyze by western blot; different parts from different membranes were delineated with dividing lines. Full-length blots are presented in Supplementary Fig. 4A **p < 0.05, *p < 0.01 or ****p < 0.001; Kruskal-Wallys test. (c) TLR4, CD47 and Sox2 expression was also determined by western blot on magnetic-isolated CD133⁺ and CD133^- Hepa129 cells cultured with pMϕ-derived CM or 4Mu-treated pMϕ-derived-CM; different parts from different membranes were delineated with dividing lines. Full-length blots are presented in Supplementary Fig. S4B, **p < 0.01CD133 + plus 4Mu-treated pMϕ-derived-CM vs. pMϕ-derived CM; 48hs Kruskal–Wallys test. (d) IL-6 production by pMϕ treated or not with 4Mu in vitro by ELISA, *p < 0.05 Unpaired T test; Data are expressed as the mean ± SEM. The in vivo and in vitro experiments were carried-out 2 times.