Fig. 1

Intervening pyruvate carboxylase stunts tumor growth by strengthening anti-tumor actions of tumor-associated macrophages. a Analysis the protein expression of gluconeogenic enzymes PCB, PCK1 and G6PC in SPMs and TAMs. b Immunoblotting of PCB in peritoneal macrophages (PM), SPMs, and TAMs isolated from tumors that grow for 1 week and 3 weeks. c Representative immunofluorescent staining of nucleus, CD14, CD163, CD68, and PCB in the human melanoma TMA. Normal tissue n=14, melanoma n=69. Scale bar, 20 μm. d Immunoblotting of PCB in macrophages treated with or without TCM under normoxic or hypoxic condition. e Quantification of PCB expression in TAMs located in HIF1α high-expressing area or HIF1α low-expressing area in tumor sections. f Lacz or PCB-OE iBMDMs were inoculated with B16 cells into the skin of the left and right hind legs of the same C57BL/6 mouse and 19 days later the size of tumors was assessed. g The percentage of CD45⁺ CD3⁺ CD8⁺ T cells among total cells in tumor digests from indicated groups. h The ratio of tumor weight between PCB-OE group and Lacz group in C57BL/6 mice, nude mice, and NSG mice. i The ratio of tumor weight between PCB-OE group and Lacz group treated with isotype IgG or anti-CD8 antibody. j The proliferation of CD8⁺ T cells cultured alone or cocultured with indicated TAMs. k The immunosuppressive genes of TAMs transfected with scramble or PCB siRNA were analyzed RT-qPCR. l T cells were co-cultured with TAMs transfected with scramble or PCB siRNA in the presence or absence of PD-1-neutralizing antibody. m Phagocytosis of B16-EGFP by TAMs was analyzed by Flow cytometry. n Cell extracts from macrophages were immunoprecipitated with anti-PCB antibody, then analyzed with anti-MYH6 and anti-PCB antibodies. o Phagocytosis of TAMs transfected with control or MYH6 siRNA were quantified by flow cytometry. p Phagocytosis of TAMs transfected with indicated siRNAs. q Co-localization of PCB, MYH6, and F-actin in TAMs was evaluated by confocal analysis. Scale bar, 10 μm. r SEM images of FDC-GTA@HSA. Scale bar, 500 nm. s The size of tumors from mice treated with PBS, GTA, FDC@HSA, or FDC-GTA@HSA. t, u The frequency of macrophages among CD45⁺ cells (t) and tumor weight (u) of indicated groups. Data represents as mean ± SEM. P < 0.05, *; P < 0.01, **; P < 0.001, ***. Significance was calculated with student’s t test and paired t test (g). Experiments were repeated at least twice to observe concordant statistical significance