Supplemental Material

This article contains the following supporting material:

• Figure 1 - Parental NMuMG cells respond to collagen I.
  Parental, uncloned NMuMG were cultured on non-coated (a), collagen I-coated (b), or fibronectin-coated (c) dishes and photographed using a 10x objective.

• Figure 2 - TGFβ is not up regulated when cells are plated on collagen I.
  (A) RT-PCR was done for TGFβ, and GAPDH (as a control). (B) NMuMG/E10 cells were cultured on non-coated or collagen I-coated dishes for 2 d in the presence of non-specific IgG (a and b) or TGFβ neutralizing antibody (c and d). (C) NMuMG/E10 cells were cultured on non-coated (a), or collagen I-coated coverslips (b) and stained for Smad 2/3. (D) NMuMG/E10 cells were infected with retrovirus encoding the inhibitory Smad, Smad7. Two d after seeding on non-coated or collagen I-coated dishes, phase contrast pictures were taken using a 10x objective.

• Figure 3 - Cdc42 is not activated when cells are plated on collagen I.
  (A) NMuMG/E10 cells were extracted 3 h after plating on non-coated, collagen I-coated or fibronectin-coated dishes. Lysates (1 mg protein) were incubated with GST-WASP coupled beads and resolved by SDS-PAGE. Cdc42-GTP and total Cdc42 were detected by Cdc42 immunoblots. (B) Cdc42N17 infected NMuMG/E10 cells were cultured for 2 d on non-coated (a) or collagen I-coated (b) dishes and photographed using a 10x objective.

• Figure 4 - Src activity is not necessary for collagen I-induced EMT.
  (A) MOCK and Src dominant negative (SrcDN) infected NMuMG/E10 cells were cultured for 2 d on non-coated (a and c) or collagen I-coated (b and d) dishes and photographed using a 10x objective. (B) Extracts (30 μg protein) from cells cultured for 2 d on non-coated or collagen I-coated dishes were resolved by SDS-PAGE and immunoblotted for N-cadherin and GAPDH.