LYL1 controls the expression of molecules involved in the formation and stabilization of adherens junctions. (A) LYL1-depletion affects the assembly and maturation of adherens junctions. hTERT1 cells were transduced with lentiviruses encoding either control shRNA or LYL1 shRNA, and puromycin-resistant cell populations were established. Confluent monolayers of the indicated population were immunostained with a VE-Cadherin antibody and vizualized with Alexa 647-conjugated secondary antibody. Note the continuous and regular staining on CTL-hTERT1 cell perimeters (arrows) and the presence of gaps between adjacent cells (arrowheads) leading to an irregular staining. (B) LYL1 depletion reduces constitutive activation of Rap1. Whole cell extract prepared from shRNA-transduced hTERT1 were assayed for RAP1 activity as described in “Rapactivity assay.” Scanned autoradiographs were quantified using Image J to determine the ration GTP-bound Rap1/total Rap1 for each extract. Bars show means ± SD of the ratio GTP-bound Rap1/total-Rap1 of 3 independent experiments. shRNA CTL-treated cell ratio was arbitrarily set at 100%. Images shown are representative of the 3 experiments. Vertical lines have been inserted to indicate a repositioned gel lane. (C) HUVECs were treated with LYL1 or control HLA-A siRNA and total RNA were prepared 48 hours after transfection. hTERT1 cells were transduced with lentiviruses encoding either control shRNA or LYL1 shRNA, and puromycin-resistant cell populations were established. mRNA levels of the indicated gene in siRNA-treated HUVEC (black bars) or in shRNA-transduced hTERT1 (gray bars) were assessed by qRT-PCR. cDNAs were amplified by specific primers and normalized to GAPDH. VE-CAD, ITGA2, Rap-GEF1, Rap-GEF2, genes encoding VE-CADHERIN, INTEGRIN-α2, C3G and DOCK4 proteins, respectively. Each bar is the mean ± SD of mRNA levels relative to control HLA-A siRNA-treated HUVECs (3 independent experiments) or to hTERT1 cells transduced with control shRNA-lentivirus, which were set at 100%. ***P < .005; **P < .01; *P < .05 by Student t test. (D) LYL1-silencing causes down-expression of INTEGRIN-α2: HUVECs or hTERT1 cells were transduced with lentiviruses encoding either control shRNA or shRNA targeting LYL1. Whole cell proteins prepared from puromycin-resistant cell populations were analyzed by immunoblotting for INTEGRIN-α2 and TAL-1 protein expression. beta-CATENIN protein expression was used to control protein loading.