Figure 3: CXCL1 secreted by mouse tumour cells attracts CXCR1-expressing ASCs.

(a) Left: RT–PCR analysis of CXCL1 messenger RNA expression in mouse ASCs and indicated mouse tumour cell lines. Data were normalized to 18S RNA. Right: CXCL1 protein concentration in medium conditioned by mouse ASCs and indicated tumour cell lines measured by ELISA. ∼ values above 5 ng ml⁻¹. (b) Immunofluorescence (IF) analysis of tumours grown in non-obese and obese mice with anti-CXCL1 antibodies indicating CXCL1 expression increased in obesity. Endothelium is counterstained with isolectin B4 (IB4). Nuclei are blue. Scale bar, 100 μm. (c) IF analysis of mouse WAT with antibodies against CXCR1 (left) and CXCR2 (right), and endothelial cell marker CD31. Note CXCR1 expression in perivascular ASCs (arrows), while CXCR2 is expressed only in circulating leukocytes and macrophage crown structures (arrowheads). Scale bar, 100 μm. (d) RT–PCR analysis of CXCR1 and CXCR2 expression in mouse ASCs and RM1 tumour cells. Data were normalized to 18S RNA. (e) Primary mouse ASCs were subjected to migration through 8-μm pores towards indicated concentrations of CXCL1 in a transwell chamber. In a,d and e, graphs show mean±s.e.m. for technical triplicates; *P<0.05 versus control (Student’s t-test). Experiments in b and c were performed for three mice with similar results; representative images are shown.