Fig 2: CDH6 and CDH17 promote the activation of ß1 and ß3 integrins. (A) Ovarian and renal cancer cell lines were exposed to RGD peptides of CDH6 or CDH17 and subjected to flow cytometry analyses to assess the activation status of ß1 and ß3 integrins. (B) SKOV-3 cells were silenced for CDH6, a2, or aIIb integrin subunits, exposed to the RGD peptides and subjected to flow cytometry assays to detect ß1 or ß3 in high-affinity conformation. (C) The same transfectants exposed to the indicated peptides were subjected to cell adhesion assays to Matrigel. (D) SKOV-3 and 786-O cells were silenced for a2 or aIIb integrin subunits and subjected to adhesion to collagen type I. Integrin activation or cell adhesion was significantly increased by the addition of RGD peptides (**P < 0.01; ***P < 0.001) or the silencing of a2 integrin subunit ($P < 0.05; $$P < 0.01) and significantly decreased by the silencing of CDH6, a2, or aIIb (?P < 0.05; ???P < 0.001), according to ANOVA tests. All results are representative of at least three independent experiments; error bars indicate standard deviation.

Fig 3: CDH6 and αIIbβ3 integrin regulate cell adhesion, migration, invasion, and proliferation in ovarian and renal cancer cells. (A) SKOV‐3 and 786‐O cells were transfected with siRNAs for the indicated genes and subjected to cell adhesion, wound healing, cell invasion, or MTT assays. Transient silencing of CDH6, αIIb, or β3 integrin subunits caused a significant decrease in cell adhesion/migratory speed/cell invasion/proliferation (*P < 0.05; **P < 0.01; ***P < 0.001), according to ANOVA tests. (B) The same transfectants were analyzed by Western blot to examine the phosphorylation status of the indicated signaling proteins. Blots were re‐probed with antibodies against the total signaling proteins as loading controls. All results are representative of at least three independent experiments; error bars indicate standard deviation.

Fig 4: Blocking cadherin‐6 disrupts thrombosis. C57BL/6J mice were treated with sheep polyclonal anti–cadherin‐6 (Cdh6, 0.8 mg/kg) 15 minutes before initiating thrombosis with FeCl3 or Rose Bengal. (A) Intravital microscope images obtained following FeCl3 treatment. In IgG‐treated mice at 8 minutes, the proximal end of the thrombus was monitored to accurately detect full occlusion. Time to stable vessel occlusion was observed and represented as Kaplan‐Meier curves for (B) FeCl3 (n = 3 per group) and (C) Rose Bengal models (n = 5 for no treatment, n = 4 for IgG or Cdh6). The curves were compared using the Mantel‐Cox log‐rank test

Fig 5: Thrombosis is delayed in CDH6 knockout mice. Thrombosis was initiated in Cdh6 -/- and wild-type mice using the (A) FeCl3 (n = 9 per group) or (B) Rose Bengal (n = 5 for wild-type and n = 6 for Cdh6-/-) models and time to vessel occlusion was observed. The vessels were monitored until a full occlusion was formed or the experiment ended and represented as Kaplan-Meier curves. The curves were compared using the Mantel-Cox log-rank test. (C) Platelet count was measured in whole blood from wild-type and Cdh6 -/- mice and compared using Student's t test. (D) Cdh6 -/- and wild-type mice were anesthetized before clipping 3 mm of tail to measure time to bleeding cessation; significance was evaluated with Student's t test, and bars represent mean ± SEM