: Supplemental

Supplemental Figure 2: Alternative oligonucleotide sequences targeted against dynamin or caveolin also block syndecan-4-induced endocytosis. Arf6 is not responsible for syndecan-4-regulated reduction in cell avidity, but mediates recycling of 5 1 integrin, related to Fig. 3. Supplemental Figure 3: An alternative oligonucleotide sequence targeted against RhoG also blocks syndecan-4-induced endocytosis, related to Fig. 5.

Primary Rhog -/-MEFs were generated by crossing heterozygous parents, isolating E13.5 embryos from knockout, wild-type and heterozygous littermates. Embryos were decapitated and eviscerated. Torsos were macerated and MEFs dissociated by 20-minute digestion in 10 mg/ml trypsin at 37 C. 5 x 10 6 MEFs were seeded into a 25-cm 2 culture dish and cultured in DME, 10% foetal bovine serum, 4.5 g/l glucose, 2 mM L-glutamine and 100 U/ml penicillin/streptomycin for up to 5 passages. Primary keratinocytes were isolated by removing skins from 1-3 day neonatal mice, and digesting with 2.5 U/ml dispaseII. The epidermis was peeled free, minced and digested with 10 mg/ml trypsin. The dissociated tissue was resuspended in KBM-Gold media (Lonza), passed through a 70-m nylon filter, and seeded onto collagen IV-coated dishes at 50000 cells/cm 2 . Cells were cultured for 2 days, before adding 10% serum to drive keratinocyte differentiation. The confluent monolayer was scratched with a pipette tip for migration analysis.

Matrix-coated surfaces
For atomic force measurement, poly-D-lysine-coated fluorodishes (World Precision Instruments) were coated with patches of 10 g/ml fibronectin (Sigma) in Dulbecco's PBS containing calcium and magnesium (Sigma) for 1 hour at room temperature, and then patches of 100 g/ml 50K at 4 C overnight. For biochemical assays and TIRF microscopy, 10-cm tissue culture-treated plastic dishes (Corning BV) or fluorodishes were coated with 10 g/ml 50K at 4 C overnight. Equivalent coating of fibronectin and 50K onto fluorodishes was tested by ELISA using the anti-fibronectin mAb 333.

Atomic Force Microscopy
Force measurements were made using a CellHesion200 atomic force head (JPK Instruments) mounted on an Olympus chassis. Cells were mounted on tipless silicon SPM-sensor cantilevers (Nanoworld) coated with 50K. The spring constant for each cantilever was calculated in situ by thermal fluctuation analysis. Cycloheximidetreated cells were resuspended in HAM's F-12 nutrient media, 25 mM HEPES, 25 g/ml cycloheximide and captured before they could adhere firmly to the dish.
Brightfield optics were used to ensure the capture of individual cells, and transfected cells were selected by epifluorescence where appropriate. Captured cells were allowed to rest for 20 minutes before taking measurements and in each case the first measurement discarded. Cantilever-mounted cells were contacted repeatedly with fluorodish coated with patches of fibronectin and 50K, at equivalent ligand density, with an applied force of 1 nN, and a 5-second contact time. Retraction measurements were made over a 90-m pulling distance with an extension/retraction speed of 5 m/s. 45-second pauses between measurements allowed the cell to recover. For stimulation experiments, baseline measurements of unbinding from 50K and fibronectin were made before injecting the soluble stimulant into the dish. Stimulants were used at the following final concentrations: H/0, 10 g/ml; H/0-heparin, 10 g/ml each; monoclonal antibodies, 10 g/ml; PMA, 30 M; MiTMAB, 100 nM (Calbiochem). Force curves were analysed using JPK data analysis software with baseline and tilt correction. For analysis of force required for individual unbinding events, steps were fitted with a significance of p < 0.001.

TIRF Microscopy
Internalisation of 1 integrin-GFP was recorded using a Leica AM TIRF system set at a 70-nm penetration depth with a 100x NA 1.47 lens. Images were acquired at 2 frames per second using a Hamamatsu C9100 EM-CCD camera. The time schedule of image acquisition was: 5 minutes on 50K; addition of H/0 or H/0 complexed with heparin (10 g/ml each); image acquisition of the stimulated cells for a further 5 minutes.

Migration Analysis
For migration over cell-derived matrices, cells were seeded at 5000 cells/ml and allowed to spread for 4 hours before capturing time-lapse images at 10-minute intervals for 10 hours on a Leica AS MDW microscope using a 5x NA 0.15 Fluotar objective and Roper CCD camera. For analysis of persistence, the migration paths of all non-dividing, non-clustered cells were tracked using ImageJ software, and persistence was determined by dividing linear displacement of a cell over 10 hours by the total distance migrated.

Western Blotting
Proteins were resolved by SDS-PAGE, transferred to nitrocellulose, and analysed using the Odyssey Western blotting fluorescent detection system (LI-COR Biosciences UK Ltd.). This involved the use of fluorophore-conjugated secondary antibodies that were detected using an infrared imaging system that allowed both an image of the membrane and an accurate count of bound protein to be recorded.

Membrane Fractionation
For each time point, 10 6 fibroblasts were spread on 50K-coated dishes in DME, 25 mM HEPES, 25 g/ml cycloheximide at 37 C for 2 hours, and then stimulated with 10 g/ml H/0 for 0-90 minutes. Cells were harvested at 4 C by scraping in Dulbecco's PBS containing calcium and magnesium, cOmplete protease inhibitor (Roche), 5 mM Na 2 VO 4 , 10 mM NaF. Membranes were fragmented by three 12-J pulses using a Vibra-Cell sonicator (Sonics), before removing nuclear debris with a 10-minute, 1000 x g centrifugation step. Membranes were separated into plasma membrane pellet and vesicle/soluble supernatant by 10-minute centrifugation at 10000 x g.
Active, precipitated GTPase was analysed by fluorescent Western blotting.
Complete cell disruption was confirmed by brightfield microscopy. Beads were isolated magnetically and washed four times with the lysis buffer, and bound protein analysed by fluorescent Western blotting.
Matrix-regulated integrin complexes were isolated from MEFs expressing 1 integrin-GFP. 2 x 10 6 cycloheximide-treated MEFs were spread on 50K-coated dishes for 2 hours, before stimulation for 10 minutes with 10 g/ml H/0 as appropriate.

Histology
Mice were sacrificed 3 days post-wounding and tissue was fixed in 4% paraformaldehyde for embedding in paraffin. 10-m sections were subjected to Masson's Trichrome staining or immunohistochemistry using -smooth muscle actin or F4/80 anti-macrophage antibody, biotinylated secondary antibody, ABC-HRP conjugate and 3,3-diaminobenzidine developing reagent (Vector Laboratories). Cells staining positively for -SMA were quantified by applying an identical, empirically determined intensity threshold to all images and counting automatically using ImageJ.