Fig. 3

Tucidinostat enhances macrophage M1 polarization in vitro and in vivo. a Relative mRNA expression of iNOS, CD86, CD206, and Arg 1 compared to vehicle (set to fold change=1) in Raw.264.7 cells and BMDMs that were exposed to increasing concentrations (2.5, 5, 7.5 μM) of tucidinostat for 24 h. The experiment was performed in triplicate. b Representative cytograms for the expression levels of MHC-II on Raw.264.7 cells and BMDMs that were exposed to increasing concentrations (2.5, 5, 7.5 μM) of tucidinostat for 24 h. c LLC cells being exposed to increasing concentrations (2.5, 5, 7.5 μM) of tucidinostat for 24 h and the tumor-conditioned medium being collected and added into Raw.264.7 cells and BMDMs for 24 h. Relative mRNA expression of iNOS, CD86, CD206, and Arg 1 compared to vehicle (set to fold change=1) in Raw.264.7 cells and BMDMs that were exposed to such tumor-conditioned medium. The experiment was performed in triplicate. d Representative cytograms for the expression levels of MHC-II on Raw.264.7 cells and BMDMs that were exposed to such tumor-conditioned medium. e Mouse CT26 cells (5 × 10⁵ cells) were engrafted into the flank of BALB/c mice. When established tumors were palpable 7 days after tumor cell inoculation, mice were treated with different doses (12.5, 25, 75 mg/kg, gavage, daily, n=5) of tucidinostat or DMSO as vehicle control (DMSO, n=5). Percentage of total macrophages (CD45⁺CD11b⁺F4/80⁺) and the percentage of M1 macrophages (CD45⁺CD11b⁺F4/80⁺/MHC-II⁺) in tumor parenchyma and tumor drainage lymph nodes from CT26 tumor-bearing mice on day 10 post-treatment initiation. The error bars indicate mean ± SEM. *P<0.05, **P<0.01, ***P<0.001 by one-way ANOVA. ns not significant, BMDM bone marrow-derived macrophage, CON control group, TCM tumor-conditioned medium, Tuc tucidinostat, iNOS inducible nitric oxide synthase, Arg 1 arginase-1