Figure 3.

sCD146 promotes BBB permeability in vitro. (A) Analysis of paracellular barrier function by permeability assay. hCMEC/D₃ cells were seeded into the upper chambers of a transwell system, and permeability was measured with 0.5 μg/mL HRP after hCMEC/D₃ cells were incubated with 5 μg/mL BSA, 0.05-5 μg/mL rhsCD146 for 2 h. *p<0.05; **p<0.01; and ***p<0.001. (B) Immunofluorescence staining of the TJPs (occludin, ZO-1 and JAM-1) and F-actin after hCMEC/D₃ cells were treated with 5 μg/mL BSA or rhsCD146 for 4 h. Bar, 10 μm. (C) hCMEC/D₃ cells were preincubated with 5 μg/mL BSA, 0.5 μg/mL or 5 μg/mL rhsCD146, TJP expression levels were verified by western blotting. (D) hCMEC/D₃ cells were treated with 5 μg/mL BSA or 0.5, 2 or 5 μg/mL rhsCD146 for 12 h, and apoptosis was detected by flow cytometry with Annexin V and 7-AAD. (E) hCMEC/D₃ cells were treated with 5 μg/mL BSA, 0.5 rhsCD146 or 5 μg/mL rhsCD146 for 12 h, and cell lysates were used to detect the expression of caspase 9, caspase 3, Bcl-2 and Bax.