Figure 6.

JCAD promotes YAP/TAZ activation in endothelial cells. (A) JCAD depletion decreased YAP/TAZ/TEAD luciferase activity (8XGTIIc-lux). Human umbilical vein endothelial cells (HUVECs) were transfected with control siRNA (siNC, 100 nM) or JCAD siRNA (siJCAD, 100 nM) for 48 h before transfection with 8XGTIIc-lux for 24 h, n = 3. YAP/TAZ siRNA (siYAP/TAZ, 20 nM) transfection was used as the positive control. (B) JCAD depletion inhibits YAP/TAZ nuclear staining in HUVECs (left panel). Quantification of YAP/TAZ subcellular localization in siNC (196 cells) and siJCAD (207 cells) treated HUVECs (right panel). C, cytoplasm; N, nucleus. Mean % from four independent experiments were used for quantification. (C) JCAD depletion decreased YAP/TAZ downstream protein expression (CTGF and Cyr61) in HUVECs, n = 3. (D) JCAD depletion promotes YAP phosphorylation at Ser127 in HUVECs, n = 3. (E) JCAD depletion decreased F-actin stress fiber formation in HUVECs. Mean fluorescence intensity was calculated by dividing total fluorescent intensity by cell numbers, n = 4. (F) JCAD interacting proteins identified by immunoprecipitation-based mass spectrometry. Averaged number of peptides from three independent experiments were presented. For a complete list of JCAD interacting proteins, please refer to Supplementary material online, Table S8. Abbreviations: ARMC8: Armadillo repeat-containing protein 8; FLNC: Filamin-C; CKAP4: Cytoskeleton-associated protein 4; PPP2R1A: Serine/threonine-protein phosphatase 2A 65 kDa regulatory subunit A alpha; TRIOBP: TRIO and F-actin-binding protein; WDR26: WD repeat-containing protein 26. (G) Validation of JCAD interaction with TRIOBP in HUVECs. HUVECs were infected with adenoviral HA-tagged (Ad-HA) JCAD for 48 h before whole cell lysate was collected for HA-beads pulldown assay, n = 3.