Integrin 9β1 mediates enhanced cell migration through nitric oxide synthase activity regulated by Src tyrosine kinase

JCS041632 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Integrin-α9β1-dependent cell adhesion. (A) SW480α9 but not SW480Mock cells, adhere to the α9-specific matrix, TnfnRAA; and the adhesion can be blocked by the α9β1 antibody (Y9A2); (n=6); **P<0.01. (B) Adhesion of SW480α9 cells to TnfnRAA in the presence or absence of Y9A2 (α9β1 antibody); inhibitors PP1 (Src), L-NAME (iNS) or ODQ (cGMP); n=3, *P<0.05, **P<0.01. DMSO-treated cells served as control. (C) Adhesion of mock transfected CHO cells on the α5β1-ligand, fibronectin (top panel), α4-tranfected CHO cells on the α4β1-ligand VCAM-1 (middle panel) or α9-transfected CHO cells on the α9β1-ligand TnfnRAA (bottom panel) in the absence or presence of specific antibodies, inhibitors: PP1 (Src), L-NAME (iNOS) or ODQ (cGMP); n=3, **P<0.01. DMSO-treated cells served as control.

• Supplemental Figure S2 -

  Fig. S2. Ligation of integrin α9β1 promotes NO and cGMP production in a Src-dependent manner. (A) Total nitrites measured from SW480α9 cell conditioned medium (MEM), when grown in the presence or absence of α9β1 specific ligand, TnfnRAA and the inhibitors, PP1 (Src) or L-NAME (iNOS); n=3, *P<0.05. (B) Concentration of cGMP measured in SW480α9 cells grown in the presence of α9-specific ligand, TnfnRAA and in the absence or presence of the inhibitors, PP1 (Src), L-NAME (iNOS) or ODQ (cGMP); n=3, **P<0.01 (as compared to the non-stimulated cells).

• Supplemental Figure S3 -

  Fig. S3. Activation of integrin α9β1 results in membrane translocation of Rac-1. SW480α9 cells suspended in serum-free medium were subsequently grown on glass cover slips with no matrix or on the α9-specific ligand TnfnRAA in the absence or presence of Src inhibitor (PP1), or iNOS inhibitor (L-NAME). The cells were then fixed, stained to detect Rac-1, and representative photomicrographs presented. Scale bar: 200µm. Arrows point out cells wherein localization of Rac-1 can be seen with clarity.

• Supplemental Figure S4 -

  Fig. S4. Integrin-α9β1-mediated iNOS phosphorylation, NO production and cell migration is FAK-independent. (A) Immunoprecipitation (with iNOS antibody) and immunoblot to detect iNOS tyrosine phosphorylation in lysates from SW480α9 cells transfected with control or FAK specific siRNA and were grown in the absence (no matrix) or presence of TnfnRAA. (B) NO concentration, as measured by Griess assay, in the conditioned medium of SW480α9 cells transfected with control (scrambled) or FAK targeting siRNA, cells cultured in the presence or absence of TnfnRAA; *P<0.05. (C) Migration of SW480α9 cells on TnfnRAA-coated transwells assessed in the absence (DMSO) or presence of inhibitors PP1, L-NAME or ODQ.

• Supplemental Figure S5 -

  Fig. S5. Src is activated with ligation of integrins α5β1, α9β1 or α4β1 western blot to detect activated Src (pY416) in lysates from CHO-mock, CHOα9 or CHOα4 cells treated with Src inhibitor (PP-1) grown for 10 minutes on the ligands fibronectin, TnfnRAA or VCAM-1, respectively.