Figure 2

Type 1 macrophages phenotype was induced by 4Mu. (a) Peritoneal macrophages (pMϕ) were isolated from healthy mice (n = 4), cultured with 0.5 mM 4Mu for 72 h (n = 2), stained with anti-F4/80, anti-CD86 and anti-CD206 antibodies, and analyzed by flow cytometry. (b) iNOS/Arg1 ratio, and cytokine gene expression were measured in pMϕ by qPCR (*p < 0.05; **p < 0.01; ****p < 0.0001 4Mu vs. saline; Mann–Whitney test and two-way ANOVA test respectively). (c) In vivo effects of 4Mu on pMϕ in mice (n = 4/group) with fibrosis-associated HCC. pMϕ were obtained on day 9 (n = 2) and 15 (n = 2). Flow cytometry of F4/80⁺ CD86⁺ and CD206⁺ cells were analyzed, and the M1/M2 proportions were calculated as log2 (CD86⁺/CD206⁺);***p < 0.001, **p < 0.01 4Mu vs. saline, on days 9 and 15, respectively; Mann–Whitney test. (d) Tumor volume was measured on 4Mu treated and non-treated tumor-bearing mice at day 9 (p = 0.0571; ns) and at day 15 (**p < 0.01; Unpaired T Test). (e) Lymphocytic infiltration of HCC tumors. Flow cytometry analysis of CD4 and CD8 T cells. Percentage of CD8⁺ cells ***p < 0.001 4Mu vs. saline, Mann–Whitney Test. Data are expressed as the mean ± SEM. Flow cytometry was analyzed using BD Accuri C6 propietary software version 1.0.264.21 (www.AccuriCytometers.com) The experiments were repeated 2 times.