Knockdown of αB-crystallin in endothelial cells inhibits tubular morphogenesis. (A) TIME cells were transfected with siRNA oligos as described in experimental procedures. Whole cell lysates were prepared 3 hours after induction of tubular morphogenesis, and the knockdown of αB-crystallin expression by siRNA was analyzed by Western blot. Actin expression was used as a loading control. (B) SiRNA-transfected cells were counted 48 hours and 96 hours after transfection. Proliferation index was calculated as the increase in total cell number compared with untransfected control (n = 3; mean ± SD). (C) Tubular morphogenesis of endothelial cells transfected with αB-crystallin–specific siRNA (CRYAB-1 and CRYAB-2) or Alexa 488–conjugated control siRNA (bar = 100 μm). (D) Quantification of tube formation (area and length) compared with control siRNA-transfected cells analyzed by the Easy Image Analysis software (n = 3; mean ± SD; **P < .05; *P < .01). (E) Whole cell extracts from siRNA-transfected cells were prepared 1, 5, or 24 hours after induction of tubular morphogenesis, and the expression of αB-crystallin and cleaved caspase-3 (17-kDa and 19-kDa fragments) were analyzed by immunoblotting. (F) αB-crystallin and cleaved caspase-3 expression was quantified and normalized to actin expression using Image Gauge software. Values are given as percentage of expression compared with the 24-hour time point of control siRNA–transfected cells (set to 100%). (G) Annexin-V (green), PI (red), and Hoechst 33 342 (blue) staining of endothelial cells transfected with αB-crystallin–specific siRNA (CRYAB-1, CRYAB-2) and unconjugated control siRNA after 24 hours of tube formation in collagen gel. (H) Quantification of apoptosis in siRNA-transfected endothelial cells after 0 hours, 12 hours, and 24 hours of tube formation. Bars represent mean values and SD of the relative apoptosis rate (fraction of annexin-V–positive, PI-negative cells normalized to control siRNA at 24 hours) of 4 independent experiments (n = 4; mean ± SD; *P < 0,05).