Figure 4
Cat S deficiency increases accumulation of autophagosomes in macrophage in the tumor microenvironment. (A) Double immunofluorescnce analysis of macrophage (anti-F4/80) and the autophagosome marker LC3 in metastatic foci from WT and Cat S-/- mice. The sections were stained with anti-F4/80 (red) or anti- LC3 (green) antibody and DAPI (blue; to stain the nuclei). Scale bars = 10 μm. (B) Double immunofluorescnce analysis of macrophage (anti-F4/80) and the autophagosome marker LC3 in WT or Cat S-/- macrophages cocultured with SL4 cells for 48 hrs and serum-starved for at least 12 hrs before co-culture. The sections were stained with anti-F4/80 (red) or anti- LC3 (green) antibody and DAPI (blue; to stain the nuclei). Scale bars = 40 μm. Three independent experiments were performed. (C) Ultrastructure was detected by transmission electron microscopy in WT or Cat S-/- macrophages cocultured with SL4 cells for 48 hrs and serum-starved for at least 12 hrs before co-culture (×10000 magnification). Three independent experiments were performed. (D) Western blot analysis of LC3 protein expression in WT or Cat S-/- macrophages cocultured with SL4 cells for 48 hrs and serum-starved for at least 12 hrs before co-culture. WT or Cat S-/- macrophages cultured individually as control group. Quantitative analysis of LC3-II/LC3-I ratio in WT or Cat S-/- macrophages cocultured with SL4 cells. Data are mean ± SEM of 3 independent experiments. **, P<0.01. NS indicates not significant.