Supplemental Material

This article contains the following supporting material:

• Supplemental Figure 1 - The presence or absence of FN in FCS does not affect FN matrix assembly in ROCK-deficient REF cells. REF were transfected with siRNAs and incubated for 2 d in normal (upper) or FN-depleted (lower) serum, then fixed and stained for FN. Bar, 50 μm.
• Supplemental Figure 2 - FN in ROCK-deficient REF cells is positive for L8 antibody staining. (A) REF were transfected with siRNAs and incubated for 2 d, then fixed and stained with L8 (green) or FN polyclonal antibodies (red). Images were acquired by laser scanning confocal microscopy. Bar, 16 μm. (B) For control experiments, REF treated with DMSO or blebbistatin overnight were also stained with L8 and FN polyclonal antibodies. Bar, 50 μm.
• Supplemental Figure 3 - Metalloprotease activities do not cause FN assembly defects in cells lacking rho kinases. REF were transfected with siRNAs and incubated in the presence of 10 μM GM6001 or DMSO for 2 d. Cultures were fixed and stained for FN. Arrowheads indicate siRNA transfected cells. Bar, 50 μm.
• Supplemental Figure 4 - ROCK-deficient REF cells express the same mRNA splice forms of FN as those of control cells. Primers corresponding to flanking regions of EIIIA, EIIIB and V regions of rat FN were used to amplify, by RT-PCR, mRNA from ROCK I (I), ROCK II (II) or control (C) siRNA treated cells. Products were resolved by agarose electrophoresis. Products that include the FN repeat are marked by arrowheads, while products resulting from an absence of the repeat are marked with double arrowheads. Both types of product were detected for EIIIA, while EIIIB insert was undetectable. Two splice forms of the V region were detected in each cell type.
• Supplemental Figure 5 - α5β1 integrin is functional in ROCK-deficient REFs. SiRNA transfected REF were fixed and stained for β1 (A) or α5 integrin (B). Bar, 50 μm. (C and D) REF transfected with Cy3-labeled siRNA were plated on plasma FN (C) or FN 110 fragment (III3-11) (D) in the absence of FCS. After 30 min, cells were fixed and stained for F-actin. Arrowheads denote siRNA transfected cells. Bar, 50 μm.