Inhibition of cANGPTL4:interacting partner complex formation delays endothelial disruption. (A,B,D,E) Immunofluorescence staining for ZO-1 (A,D) and TER measurement (B,E) in a confluent HMVEC monolayer. Cells were treated with either 6 μg/mL cANGPTL4 in the presence of increasing anti-integrin β1 concentrations (1:100 and 1:50; A-B) or 6 μg/mL cANGPTL4 followed by removal of exogenous cANGPTL4 at 90 minutes (D,E). Treatments were for 3 or 6 hours. 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. Data (means ± SD) from 3 independent experiments. *P < .05; **P < .01. (C) In situ kinetic PLA detection of cANGPTL4: indicated binding partner complexes in cANGPTL4-treated HMVECs in the presence of anti-integrin β1. Values (means ± SD) represent mean fold change in the number of interactions compared with the zero time point, as determined from n = 3 independent experiments (∼ 600 HMVECs) using BlobFinder. Experiments were terminated when microscopic lesions were observed (360 minutes after cANGPTL4 treatment).