Figure 6. Validation of CD31 as a YAP-dependent regulator of redox functions in angiosarcoma cells.

(A) CD31^high cells lost their tube forming capacity after treatment with an anti-CD31 blocking monoclonal antibody (CD31 mAb) for 18 hours in a concentration-dependent fashion. (B) Treatment with CD31 mAb for 24 hours effectively reduced basal oxidative stress levels (indicated by flow cytometric DCF staining) selectively in CD31^high cells, while similar treatment did not alter ROS level in CD31^low cells. (C) Immunoblot anaylsis demonstrated that CD31 mAb treatment in CD31^high cells under 1% serum-starvation induced YAP expression that was paralleled by decreased levels of γ-H2AX, indicating attenuated basal oxidative DNA damage. Medium with or without CD31 mAb was replaced every 24 hours. (D) CD31^high cells were treated either with selective siRNA against CD31 (siRNA_CD31) or scrambled sequence (scr_siRNA). Immunoblot analysis was performed 48 hours after transfection showing selective knockdown of CD31, together with a marked increase of YAP and suppression of oxidative DNA, indicated by γ-H2AX. (E) Flow cytometric quantification of DCF fluorescence at the same time point showed a significant increase of endogenous oxidative stress levels in YAP-depleted CD31^low cells compared to scrambled transfected controls (n=3). (F) Selective knockdown of YAP (siRNA_YAP) in CD31^low cells resulted in a marked induction of γ-H2AX compared to scrambled transfected controls (scr_siRNA), 48 hours after transfection. (G) Flow cytometric quantification of DCF fluorescence at the same time point showed a significant increase of endogenous oxidative stress levels in YAP-depleted CD31^low cells compared to scrambled transfected controls (n=3). All data are mean ± SEM and were analyzed using either two-way ANOVA (B) or unpaired t-test (E, G) (*p < 0.05).