Journal: Molecular Oncology

Article Title: CDH6‐activated αIIbβ3 crosstalks with α2β1 to trigger cellular adhesion and invasion in metastatic ovarian and renal cancers

doi: 10.1002/1878-0261.12947

Figure Lengend Snippet: Expression of RGD Cadherins and integrins in renal and ovarian cancer cells. (A) Western blot analysis of the expression of CDH6, CDH17, and the indicated integrin subunits in renal cancer, ovarian cancer, and colorectal cancer cells. β‐Tubulin expression was used as loading control. (B) Flow cytometry analysis of cadherin and integrin expression in the indicated cell lines. (C) Changes in the expression of the indicated cadherins and integrin subunits as a function of SKOV‐3 cell confluency by flow cytometry (left) and Western blot analysis (right). All results are representative of at least three independent experiments; error bars indicate standard deviation.

Article Snippet: CDH6 antibody (M06353) for immunofluorescence was from Boster (Pleasanton, CA, USA), whereas antibodies for Src (AF3389) and CDH6 (AF2715) for Western blot and flow cytometry were from R&D Systems (Minneapolis, MN, USA). β‐Tubulin antibody (ab21057) and phospho‐focal adhesion kinase (611722) were from Abcam (Cambridge, UK) and BD Transduction Laboratories (Franklin Lakes, NJ, USA), respectively. β1 and β3 integrin‐specific antibodies for high‐affinity conformation were from BD Biosciences, San Diego, CA, USA (HUTS21) and Merck, Kenilworth, NJ, USA (MABT27), respectively.

Techniques: Expressing, Western Blot, Control, Flow Cytometry, Standard Deviation

Journal: Molecular Oncology

Article Title: CDH6‐activated αIIbβ3 crosstalks with α2β1 to trigger cellular adhesion and invasion in metastatic ovarian and renal cancers

doi: 10.1002/1878-0261.12947

Figure Lengend Snippet: CDH6 binds αIIbβ3 or α2β1 integrins in different cell lines. (A, B, C) The indicated cells were immunoprecipitated using anti‐CDH6, anti‐CDH17, anti‐αIIb, or control antibodies. Immunoprecipitates were analyzed by Western blot using the indicated antibodies. (D) SKOV‐3 and OVCAR3 cells were used to determine CDH6, α2, and αIIb integrin subunits colocalization by confocal microscopy. Pearson’s correlation coefficient was calculated for each colocalization experiment (right). Bar size: 20 µm.

Article Snippet: CDH6 antibody (M06353) for immunofluorescence was from Boster (Pleasanton, CA, USA), whereas antibodies for Src (AF3389) and CDH6 (AF2715) for Western blot and flow cytometry were from R&D Systems (Minneapolis, MN, USA). β‐Tubulin antibody (ab21057) and phospho‐focal adhesion kinase (611722) were from Abcam (Cambridge, UK) and BD Transduction Laboratories (Franklin Lakes, NJ, USA), respectively. β1 and β3 integrin‐specific antibodies for high‐affinity conformation were from BD Biosciences, San Diego, CA, USA (HUTS21) and Merck, Kenilworth, NJ, USA (MABT27), respectively.

Techniques: Immunoprecipitation, Control, Western Blot, Confocal Microscopy

Journal: Molecular Oncology

Article Title: CDH6‐activated αIIbβ3 crosstalks with α2β1 to trigger cellular adhesion and invasion in metastatic ovarian and renal cancers

doi: 10.1002/1878-0261.12947

Figure Lengend Snippet: 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.

Article Snippet: CDH6 antibody (M06353) for immunofluorescence was from Boster (Pleasanton, CA, USA), whereas antibodies for Src (AF3389) and CDH6 (AF2715) for Western blot and flow cytometry were from R&D Systems (Minneapolis, MN, USA). β‐Tubulin antibody (ab21057) and phospho‐focal adhesion kinase (611722) were from Abcam (Cambridge, UK) and BD Transduction Laboratories (Franklin Lakes, NJ, USA), respectively. β1 and β3 integrin‐specific antibodies for high‐affinity conformation were from BD Biosciences, San Diego, CA, USA (HUTS21) and Merck, Kenilworth, NJ, USA (MABT27), respectively.

Techniques: Migration, Transfection, Western Blot, Phospho-proteomics, Standard Deviation

Journal: Molecular Oncology

Article Title: CDH6‐activated αIIbβ3 crosstalks with α2β1 to trigger cellular adhesion and invasion in metastatic ovarian and renal cancers

doi: 10.1002/1878-0261.12947

Figure Lengend Snippet: CDH6 and CDH17 promote the activation of β1 and β3 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 β1 and β3 integrins. (B) SKOV‐3 cells were silenced for CDH6, α2, or αIIb integrin subunits, exposed to the RGD peptides and subjected to flow cytometry assays to detect β1 or β3 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 α2 or αIIb 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 α2 integrin subunit ($ P < 0.05; $$ P < 0.01) and significantly decreased by the silencing of CDH6, α2, or αIIb (◊ 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.

Article Snippet: CDH6 antibody (M06353) for immunofluorescence was from Boster (Pleasanton, CA, USA), whereas antibodies for Src (AF3389) and CDH6 (AF2715) for Western blot and flow cytometry were from R&D Systems (Minneapolis, MN, USA). β‐Tubulin antibody (ab21057) and phospho‐focal adhesion kinase (611722) were from Abcam (Cambridge, UK) and BD Transduction Laboratories (Franklin Lakes, NJ, USA), respectively. β1 and β3 integrin‐specific antibodies for high‐affinity conformation were from BD Biosciences, San Diego, CA, USA (HUTS21) and Merck, Kenilworth, NJ, USA (MABT27), respectively.

Techniques: Activation Assay, Flow Cytometry, Standard Deviation

Journal: Molecular Oncology

Article Title: CDH6‐activated αIIbβ3 crosstalks with α2β1 to trigger cellular adhesion and invasion in metastatic ovarian and renal cancers

doi: 10.1002/1878-0261.12947

Figure Lengend Snippet: CDH6 and αIIb/α2 integrins are required for lung homing in ovarian and renal cancer. (A) RNA was isolated from liver or lungs of mice inoculated with SKOV‐3 cells previously silenced for the indicated genes and subjected to RT‐PCR assays to detect human GAPDH as surrogate of cell colonization. Murine β‐actin was used as loading control. (B) Model of integrin crosstalk. According to our results, the presence of CDH6 activates αIIbβ3 integrin, which induces the activation of α2β1 integrin. The activation of integrins promotes cell adhesion, invasion, and proliferation, leading to the metastatic dissemination of cancer cells.

Article Snippet: CDH6 antibody (M06353) for immunofluorescence was from Boster (Pleasanton, CA, USA), whereas antibodies for Src (AF3389) and CDH6 (AF2715) for Western blot and flow cytometry were from R&D Systems (Minneapolis, MN, USA). β‐Tubulin antibody (ab21057) and phospho‐focal adhesion kinase (611722) were from Abcam (Cambridge, UK) and BD Transduction Laboratories (Franklin Lakes, NJ, USA), respectively. β1 and β3 integrin‐specific antibodies for high‐affinity conformation were from BD Biosciences, San Diego, CA, USA (HUTS21) and Merck, Kenilworth, NJ, USA (MABT27), respectively.

Techniques: Isolation, Reverse Transcription Polymerase Chain Reaction, Control, Activation Assay