Supplemental Material

This article contains the following supporting material:

• Figure 1 - Interaction of cell surface tTG with immobilized or soluble 42 kDa fragment of fibronectin activates RhoA. (A) Concentration dependence of RhoA activation by adhesion of NIH3T3-tTG transfectants on the 42 kDa fibronectin fragment. Cells were plated for 15 min in serum-free medium on tissue culture plates coated with different concentrations of the 42 kDa fragment. (B) Time dependence of RhoA activation by adhesion of NIH3T3-tTG transfectants on the 42 kDa fibronectin fragment. Cells were plated for indicated periods of time in serum-free medium on tissue culture plates coated with 20 μg/ml of the 42 kDa fragment. (C) The 42 kDa fragment of fibronectin was cross-linked in solution with 20 mM DSP (Dithiobis-[succinimidylpropionate], Pierce, Rockford, IL). The resulting DSP-crosslinked oligomers were separated from the monomeric 42 kDa fragment by FPLC using Superdex 75 (10x300) column and purity of the fractions was analyzed by SDS-PAGE. Positions of molecular mass markers (left lane) are assigned. (D) Time course of RhoA activation in NIH3T3-tTG transfectants in suspension by soluble 42 kDa monomer and oligomers. The numbers beneath the RhoA-GTP bands display normalized intensities compared to the value of 1.0 for cells in suspension (A,B) or in untreated cells (D).

• Figure 2 - Down-regulation of Src enzymatic activity is involved in RhoA activation by the interaction of cell surface tTG with the 42 kDa fragment of fibronectin. (A) Adhesion of NIH3T3-tTG transfectants on the 42 kDa fragment decreases Src kinase activity. Src kinase activity in cell lysates was determined by detection of Tyr phosphorylation of SignalScout ™ GST-Src kinase substrate (Stratagene). NIH3T3-vector and NIH3T3-tTG transfectants were either held in suspension or plated on the 42 kDa fibronectin fragment for 15 min. The relative activities of Src kinase were compared to that in NIH3T3-vector transfectants in suspension, which was expressed as 100%. (B) Inhibition of Src kinase in NIH3T3-tTG transfectants adherent to the 42 kDa fibronectin fragment is involved in upregulation of RhoA activity. Both types of transfectants were either left untreated or were treated with Src kinase inhibitor PP2 and then plated on the 42 kDa fragment of fibronectin for 15 min. The levels of RhoA-GTP in the transfectants were normalized for total RhoA loadings and then converted to the percentages of active RhoA in untreated and PP2-treated cells. Note that PP2 treatment of cells on the 42 kDa fragment raises sharply the activation level of RhoA in NIH3T3-vector transfectants, but increases it only moderately in NIH3T3-tTG cells.