Figure 2: Smad3 facilitates cancer progression by suppressing host NK cell immunity in the tumour microenvironment.

(a) Immunofluorescence detects the tumour-infiltrating NK1.1⁺, NKp46⁺ and NK1.1⁺ INF-γ⁺ NK cells in the B16F10 tumour collected on day 7. Representative images of tumour sections stained with the antibodies recognizing NK1.1 (green, upper panel), NKp46 (green, middle panel), NK1.1 (red) and IFN-γ (green, lower panel) are shown. Nuclei were counterstained with DAPI (blue), and the percentage of positive cells in the tumour tissues of Smad3^−/− or Smad3^+/+ mice are shown (right panel). (b) Two-colour flow cytometry shows the population of tumour-infiltrating NK1.1⁺ CD49b⁺ cells in the B16F10 tumour. (c) Western blotting analysis detects the NKp46 expression within the tumour tissues. (d,e) Enzyme-linked immunosorbent assay analysis determines the levels of granzyme B, IL-2 and IFN-γ in the tumour tissues (d) and circulation (e). (f) Effects of NK cell depletion on cancer progression in B16F10 tumour-bearing Smad3^−/− mice as determined by bioluminescent imaging, tumour volume measurement and the survival rate. Data represent mean±s.d. for groups of 3–5 mice. *P<0.05, **P<0.01 compared with B16F10 tumour-bearing Smad3^+/+ (a–e) or Smad3^−/− mice (f) analysed by analysis of variance. Scale bars, 50 (upper panel in a) and 100 μm.