Figure 4

CX3CR1 deficiency increases macrophage apoptosis in metastasized tumor. (A) The TUNEL assay and immunofluorescence analysis of macrophage apoptosis from metastasized foci after intrasplenic injection of SL4 cells in WT and CX3CR1^−/− mice. Representative micrographs show F4/80-positive (red), TUNEL-positive signal (green), nuclei (blue) in metastatic foci. The apoptotic index of macrophages was assessed as the percentage of apoptotic TUNEL⁺F4/80⁺ cells to the total number of F4/80⁺ cells. Scale bars =40 μm. Data are mean ± SEM for n = 8 mice with 10 fields per animal. **, P<0.01. (B) Western blot analysis of the protein levels of Bax, Bcl-2, phospho-FOXO3a, FOXO3a and activate caspase3 from metastasized foci in the liver after intrasplenic injection of SL4 cells in WT and CX3CR1^−/− mice. GAPDH was used as a loading control. Quantitative analysis of Bax/ Bcl-2 ratio, phospho-FOXO3a/FOXO3a ratio and activate caspase3 expression in metastasized foci from WT and CX3CR1^−/− mice. Data are mean ± SEM for n = 8 mice. **, P<0.01. NS, not significant. (C) WT or CX3CR1^−/− macrophages were co-cultured with SL4 cells in peptide gels and then underwent TUNEL assay and immunofluorescence analysis of macrophage apoptosis by staining with F4/80 antibody. The nucleus was stained with DAPI. Representative micrographs show F4/80-positive (red), TUNEL-positive signal (green), nuclei (blue) in coculture. The apoptotic index of macrophages was assessed as the percentage of apoptotic TUNEL⁺F4/80⁺ cells to the total number of F4/80⁺ cells from 10 randomly selected regions. Scale bars = 25 μm. Data are mean ± SEM of 3 independent experiments. **, P<0.01. Mφ indicates macrophages.