Fig. 6

smTRAIL exerts direct effects on macrophages in vitro. A M0, M1, and M2-like macrophage cell morphologies by immunofluorescence microscopy. Different concentrations of protein were incubated with macrophages for 24 h, and the cell morphology was determined by CD11b and DAPI staining. Scale bar, 10 μm. B The levels of Il-6 mRNA and Il-10 mRNA in M0, M1, and M2-like macrophages after smTRAIL incubation analyzed by real-time RT-PCR and normalized to β-actin (n = 3). C The percentages of CD206⁺ M2-like macrophages (red frame) and CD86⁺CD206⁺ M2-like macrophages (blue frame) were detected by flow cytometry. D The percentage of M2b-like macrophages (IL-12⁻ IL-10⁺ macrophages) in CT26 tumor tissues was detected by flow cytometry (n = 5). E Levels of mRNA expression of Ccl1 in CT26 were analyzed by real-time RT-PCR and normalized to β-actin (n = 5). F FoxO1 expression in macrophages by western blot. After incubating M0 and M2-like macrophages for 24 h with different protein levels, the cells were harvested, and the expression of FoxO1 was detected by SDS-PAGE followed by western blotting with (C29H4) rabbit mAb against FoxO1. G Western blot of FoxO1 expression after silencing of the FoxO1 gene in macrophages. H The level of Il-10 mRNA was analyzed by real-time RT-PCR and normalized to β-actin after silencing of the FoxO1 gene in M0 and M2-like macrophages (n = 3). I IL-10 expression analyzed by ELISA after silencing of the FoxO1 gene in M0 and M2-like macrophages (n = 3). J After FoxO1 silencing, M2-like macrophages were incubated with and without smTRAIL for 24 h. The percentage of CD206⁺ M2-like macrophages was determined by flow cytometry. K Mechanism of smTRAIL acting on macrophages. A one-way ANOVA test was performed to calculate the significant differences between groups, followed by LSD analysis. *P < 0.05; **P < 0.01; ***P < 0.001; and ****P < 0.0001