Fig. 5

Knockdown of Beclin1 inhibits tumor growth and alters TAMs polarization. a BECN1-KD B16F10 and Ctrl-B16F10 cells were s.c. implanted to C57BL/6 mice (n = 6 per group). Tumor area was measured at the described days. b Tumor bearing mice were euthanized around day 15, total TAMs (F4/80⁺ CD11b⁺) were gated and analyzed for mean fluorescent intensity (MFI) of CD206, PD-L1, CD86 and MHC II expression by flow cytometry. Events are gated on singlet, live and CD45⁺ tumor infiltrating cells. c TILs were stimulated with Cell Stimulation Cocktail for 5 h, the frequency of CD4⁺ IFN-γ⁺ and CD8⁺ IFN-γ⁺ T cells was determined by intracellular staining. Single suspension cells from dLNs and spleens were stimulated with mitomycin C inactivated B16F10 cells at a ratio of 30:1 for 21 h, breferdin A plus monensin was added for the last 5 h of culture, and IFN-γ production was determined by flow cytometry. d T cells from TILs, dLNs and spleens were tested for Ki-67 expression by intracellular staining. e B16F10 cells were mixed with WT or PD-L1^−/− BMDMs (2:1) treated with or without TRAPs and then injected s.c. to C57BL/6 mice (n = 6 per group). Tumor growth was monitored at the indicated days. Data (mean ± SEM) are representative of three independent experiments. *p < 0.05 and ***p < 0.001 by unpaired t test (a-d) and two-way ANOVA (e)