Fig 2: CLEC11A-expressing tumors contain more CD31-positive cells of endothelial lineage. (A) Tissue sections prepared from tumors formed by H1299-Vec and H1299-CLEC11A cells were subjected to IHC staining using an anti-CD31 antibody. (B) Images from the experiments of panel A were quantitated and the data were analyzed by Student t-test. (C) In vivo angiogenesis assays were performed as described in Materials and Methods. The levels of blood vessel content in Matrigel with FGF1 or FGF1 plus CLEC11A were quantitated by measuring the hemoglobin (Hb) levels. The symbols (open and closed) with the same shape represented the data collected from Matrigel blobs of the same mouse. (D) This experiment was performed as described in panel C, except using VEGF-A instead of FGF1. The data were presented as the ratios of Hb content in the Matrigel blob containing VEGF plus CLEC11A over that containing VEGF alone from the same mouse.

Fig 3: CLEC11A expression is frequently detected in LAC tissues with an EGFR mutation. Tissue sections from LAC with or without EGFR kinase domain mutations were examined by IHC staining using an anti-CLEC11A monoclonal antibody. The light staining patterns of the connective tissue within the tumor sections served as internal, negative controls. Scale bar: 0.2 mm.

Fig 4: CLEC11A is a factor with angiogenic potential. (A) The chemo-attractant ability of purified CLEC11A (30 ng/mL) was tested against SVEC4-10 cells in the presence or absence of the anti-CLEC11A monoclonal antibody (1 μg/mL) in the Transwell chambers. (B) The chemo-attractant ability of purified CLEC11A (30 ng/mL) was tested against primary AAE cells in the Transwell chambers. (C) Conditioned media of HCC827-Vec and HCC827-shCLEC11A cells were tested for their chemo-attractant ability against SVEC4-10 cells. (D) Tissue sections prepared from tumors formed by HCC827-Vec and HCC827-shCLEC11A cells were subjected to IHC staining using an anti-CD31 antibody. Staining images were quantitated and the data were analyzed by the Student t-test. (E) Overnight 1% serum-starved SVEC4-10 cells were treated with 30 ng/mL of CLEC11A for the indicated times and the levels proteins of interest were examined by immune-blotting. (F) The chemo-attractant activities of CLEC11A (30 ng/mL) or VEGF-A (20 ng/mL) were tested against SVEC4-10 cells in the presence or absence of vatalanib (5 mM) in the Transwell chambers. Statistical significances between treatments are presented as single asterisk (p < 0.05) or double asterisks (p < 0.01).

Fig 5: Suppression of CLEC11A expression in HCC827 cells decreases tumorigenic ability. (A) HCC827-Vec and HCC827-shCLEC11A cells were established by permanent transfection of pSuper-puromycin and pSuper-shCLEC11A vectors, respectively. The expression levels of the indicated proteins in the conditioned medium (CM) were examined by immune-blotting. (B) HCC827-Vec and HCC827-shCLEC11A cells were injected subcutaneously into nude mice. Tumors were harvested 5 weeks post-transplantation, weighed, and photographed. (C) Tumors formed by HCC827-shCLEC11A cells were significantly larger (Student t-test) than those formed by control cells.