Figure 6.

Dlx2 is required for B16 melanoma primary tumour growth and lung metastasis. (A) Dlx2 expression is induced by TGFβ in melanoma cells. B16 melanoma cells were treated with TGFβ for 6 days and Dlx2 mRNA levels were determined by quantitative RT–PCR. Values were normalized to endogenous RPL19 mRNA levels. (B) Reduced primary tumour growth in B16 melanoma cells transfected to stably express shRNA against Dlx2. Three independent Dlx2-specific shRNA sequences (shDlx2 1–3) and one control shRNA sequence (shCTR) were used to establish stable cell pools. Cells were injected into both flanks of 9–10 C57Bl/6 mice per cell pool and tumour weights were measured 2 weeks after implantation. (C) Reduced metastatic outgrowth of B16 melanoma cells depleted for Dlx2 expression. Micrometastatic lesions were counted on histological sections (shown in D) of the lungs of the mice described in (B) (five lungs per cell pool were analysed). (D) Serial histological sections of lungs from C57/Bl6 injected subcutaneously with shDlx2-1 and shCTR B16 melanomas cells were stained with haematoxylin/eosin. Scale bar=100 μm. (E) Tumour sections were stained against cleaved caspase 3 to quantify the rate of apoptosis. The moderate increase in apoptosis observed in Dlx2-depleted tumours was not statistically significant. Data are shown as mean±s.d. Statistical values are calculated by using an unpaired, two-tailed t-test. ^*P⩽0.05; ^**P⩽0.01; ^***P⩽0.001.