cANGPTL4 is elevated in metastatic tumors and disrupts endothelial junction integrity. (A-B) Expression levels of cANGPTL4 mRNA (A) and protein (B) in basal cell carcinoma (BCCs) and metastatic melanoma paired with peritumor normal tissues as determined by quantitative real-time PCR and immunoblot, respectively (n = 3). Data spots from same individual are linked by colored lines. Ribosomal protein L27 (L27) was used as a reference housekeeping gene. β-tubulin served as a loading and transfer control. (C-D) Evans blue dye extravasation induced by either A-5RT3_CTRL– or A-5RT3_ΔANGPTL4–induced tumors of different (C) or similar (D) tumor volume (A-5RT3_CTRL, 1098 ± 422 mm³ vs A-5RT3_ΔANGPTL4, 551 ± 135 mm³; P < .05; n = 6). (E-F) Evans blue dye extravasation induced by cANGPTL4 or PBS vehicle (G) and quantification (H) of extravasated Evans blue dye by measurement of absorbance at 610 nm. *P < .05. (G) Immunodetection of CD31 in A-5RT3_CTRL– and A-5RT3_ΔANGPTL4–induced tumors. (H) Immunofluorescence staining for CD31 and cANGPTL4 in B16F10_CTRL- and B16F10_ΔANGPTL4-induced tumors. Scale bar represents 100 μm. (I) Immunofluorescence staining for ZO-1 in a confluent HMVEC monolayer. Cells were treated with either CM from A-5RT3_CTRL in the presence or absence of anti-cANGPTL4, from A-5RT3_ΔANGPTL4 cells. HMVECs were counterstained with DAPI (blue) for nuclei and phalloidin (red) for actin stress fibers. Scale bar represents 40 μm. White arrows indicate endothelial junction lesions. (J) Transendothelial electrical resistance (TER) analysis of confluent monolayer HMVECs treated with the indicated amounts of cANGPTL4. PBS served as a vehicle control. Data (means ± SD) from 3 independent experiments.