American Society of Hematology

Figure 3

$Figure 3. CD151 is needed for proper endothelial cell-cell adhesion. (A) Cell aggregation assay. A total of 2 × 104 cells were seeded into 30-μL hanging drop cultures in either complete or Ca2+-depleted media and allowed to aggregate at 37°C overnight. After passing the cell cluster 10 times through a 200-μL pipette tip, cell clusters were imaged, and the degree of dissociation of the aggregates was quantified using ImageJ 1.42q (published by Wayne Rasband, National Institutes of Health) software analysis. Representative images of aggregation in untreated cells (left panel) and the quantitative results (right panel). Bar represents 100 μm. *P < .01. (B) AJ complexes are mislocalized in CD151-silenced ECs. HUVEC transductants were cultured for 4 days to confluence. After 6-hour starvation, HUVEC monolayers were fixed, permeabilized, and stained with specific Abs. The distributions of VE-cadherin and β-catenin were visualized using a Zeiss LSM510 confocal fluorescence microscope under a 100×/1.4 NA oil objective. Bar represents 10 μm. (C) Deficient formation and maturation of adhesion zipper on CD151 silencing. HMEC transductants were fixed without permeabilization, probed with CD9 mAb and AlexaFluro-488–conjugated secondary Ab, and visualized by TIRF microscopy. Bar represents 15 μm. (D) Abnormal endothelial cell-cell adhesion in lung vessels of CD151 KO mice. Twelve-week-old male CD151 KO (n = 4) and littermate WT (n = 4) mice were perfused and fixed with 2.5% glutaraldehyde. Mouse lung tissue was excised and processed for transmission electron microscopy. Bar represents 100 nm. Right panel: Abnormal EC junctions were counted visually. *P < .01. (E) The protein association in AJ complexes is not affected by the loss of CD151. HMEC-MOCK or HMEC-CD151 cells were lysed in coimmunoprecipitated buffer. The indicated proteins were immunoprecipitated, followed by immunoblotting with specific antibodies. (F) Detergent solubility assay of VE-cadherin. HMEC-MOCK or HMEC-CD151 KD cells were solubilized with 0.15% Triton X-100. Soluble and insoluble fractions were obtained after ultracentrifugation, subjected to SDS-PAGE, followed by immunoblotting with anti–VE-cadherin mAb.$

CD151 is needed for proper endothelial cell-cell adhesion. (A) Cell aggregation assay. A total of 2 × 10⁴ cells were seeded into 30-μL hanging drop cultures in either complete or Ca²⁺-depleted media and allowed to aggregate at 37°C overnight. After passing the cell cluster 10 times through a 200-μL pipette tip, cell clusters were imaged, and the degree of dissociation of the aggregates was quantified using ImageJ 1.42q (published by Wayne Rasband, National Institutes of Health) software analysis. Representative images of aggregation in untreated cells (left panel) and the quantitative results (right panel). Bar represents 100 μm. *P < .01. (B) AJ complexes are mislocalized in CD151-silenced ECs. HUVEC transductants were cultured for 4 days to confluence. After 6-hour starvation, HUVEC monolayers were fixed, permeabilized, and stained with specific Abs. The distributions of VE-cadherin and β-catenin were visualized using a Zeiss LSM510 confocal fluorescence microscope under a 100×/1.4 NA oil objective. Bar represents 10 μm. (C) Deficient formation and maturation of adhesion zipper on CD151 silencing. HMEC transductants were fixed without permeabilization, probed with CD9 mAb and AlexaFluro-488–conjugated secondary Ab, and visualized by TIRF microscopy. Bar represents 15 μm. (D) Abnormal endothelial cell-cell adhesion in lung vessels of CD151 KO mice. Twelve-week-old male CD151 KO (n = 4) and littermate WT (n = 4) mice were perfused and fixed with 2.5% glutaraldehyde. Mouse lung tissue was excised and processed for transmission electron microscopy. Bar represents 100 nm. Right panel: Abnormal EC junctions were counted visually. *P < .01. (E) The protein association in AJ complexes is not affected by the loss of CD151. HMEC-MOCK or HMEC-CD151 cells were lysed in coimmunoprecipitated buffer. The indicated proteins were immunoprecipitated, followed by immunoblotting with specific antibodies. (F) Detergent solubility assay of VE-cadherin. HMEC-MOCK or HMEC-CD151 KD cells were solubilized with 0.15% Triton X-100. Soluble and insoluble fractions were obtained after ultracentrifugation, subjected to SDS-PAGE, followed by immunoblotting with anti–VE-cadherin mAb.

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