Figure 6.

Wnt5a-triggered control of hCMEC/D3 permeability is mediated by Par/aPKC planar cell polarity complex. (A) HCMEC/D3 cells were incubated with EBM-2 medium or with EBM-2 containing 10 mmol/L lithium chloride (LiCl), 40% of control (CM CTR) or Wnt5a (CM 5a)-conditioned medium and grown at confluence for 6 days on culture inserts. Total RNA extracts were obtained using RNA kit extraction. Reverse transcription was performed and Axin2 messenger RNA expression was evaluated by quantitative real time polymerase chain reaction using LightCycler 480 device. Results are visualized as relative expression compared with EBM-2 medium. (B) Immunoblot analysis of JNK phosphorylation and actin expression. HCMEC/D3 cells grown at confluence for 6 days were treated for 30 minutes or 1 hour with EBM-2 completed with 40% of CM CTR or CM 5a. Whole-cell lysates were generated. Proteins were separated on sodium dodecyl sulfate polyacrylamide gel electrophoresis gel followed by immunoblotting with anti-Phospho-JNK polyclonal antibody, anti-JNK monoclonal antibody or anti-actin monoclonal antibody. (C) HCMEC/D3 cells were incubated with EBM-2 medium or with EBM-2 containing 10 mmol/L LiCl, 40% of CM CTR or CM 5a-conditioned medium and grown at confluence for 6 days on culture inserts. Permeability assays to Lucifer Yellow (LY) were performed and the permeability coefficient was quantified. Results are expressed as permeability ratio, normalized relative to the permeability coefficient for EBM-2 cultured cells. The basal permeability level was 1.8±0.1.10⁻³ cm/minute. Results are means±s.d. for seven independent experiments. Indicated P values were obtained using Student's t-test. *P<0.01; **P<0.003, ***P<0.002. (D) HCMEC/D3 cells were treated with EBM-2 complement with 40% of CM CTR or CM 5a and grown at confluence for 6 days on culture inserts. Then cells were fixed and permeabilized. Immunofluorescence labeling was performed with a PAR-3 polyclonal antibody. (E) HCMEC/D3 cells were treated with EBM-2 completed with 40% of CM CTR or CM 5a and grown at confluence for 6 days on culture inserts. Mean apical/basal fluorescence intensity of 40 different cells from at least three independent experiments is shown. Indicated P value was obtained using Student's t-test: *P<0.0001. (F) HCMEC/D3 cells treated with control siRNA or PAR-3 siRNA (#A) were grown at confluence for 6 days in EBM-2 medium completed with 40% of CM CTR or CM 5a on culture inserts. Permeability assays to LY were performed and the permeability coefficient was quantified. Results are expressed as permeability ratio, normalized relative to the permeability coefficient for CM CTR. The basal permeability level was 1.65±0.2.10⁻³ cm/minute. Results are means±s.d. for five independent experiments. Indicated P values were obtained using Student's t-test. *P<0.02; NS, not significant.