cANGPTL4:interacting partner complex formation triggers the nuclear translocation of β-catenin. (A) Immunofluorescence staining for β-catenin (green) in an HMVEC monolayer treated with either 6 μg/mL cANGPTL4 or 2.5mM EDTA (as positive control) for 3 hours. HMVECs were counterstained with DAPI (blue) for nuclei. Scale bar represents 40 μm. White arrows indicate endothelial junction lesions associated with reduced β-catenin staining. Representative fluorescence intensity plot of β-catenin and DAPI, indicated by the white dotted line across the nuclei, were quantified using ZEN 2009 software (Carl Zeiss). (B) In situ PLA for β-catenin and quantification of the number of PLA spots per nuclei of cANGPTL4-treated HMVECs at the indicated time points. (0 minutes, 1.2 ± 0.81; 40 minutes, 3 ± 1.23; 90 minutes, 7.9 ± 1.93; 140 minutes, 13.3 ± 2.44; 180 minutes, 17.5 ± 2.68) PLA signals (red) revealed translocation of β-catenin into nuclei at 140 minutes after treatment (∼ 250 HMVECs). Cells were counterstained with Hoechst dye for nuclei (blue). Scale bar represents 40 μm. *P < .05; **P < .01. (C) Immunodetection of β-catenin in membrane, cytosol, and nuclei fractions of HMVECs treated with either vehicle (PBS) or cANGPTL4. Protein lysates were collected at the indicated times. Values (means ± SD) below each band represent the mean fold change in protein expression level compared with the cognate zero time point (n = 3). *P < .05; **P < .01. (D) Schematic diagram of cANGPTL4-mediated disruption of endothelial junctions. (1) cANGPTL4:integrin α5β1 formation (30-50 minutes) coincides with (2) the activation of Rac-GTP and pPAK in ECs (40-60 minutes) and with vascular leakiness (3); the interaction between cANGPTL4 with VE-cadherin and claudin-5 (120-140 minutes) disrupts intercellular contact formation, and stimulates (4) nuclear translocation of β-catenin (180 minutes).