JCS020941 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Adhesion organization in cells knocked down for the expression of FAK, PDZ-RhoGEF, ROCK I and ROCKII. Control cells, FAK-, PDZ-RhoGEF-, ROCKI- and ROCKII-knockdown cells were plated on coverslips coated with 1 µg/ml fibronectin overnight. Cells were fixed and immunostained for FAK and paxillin as described in Materials and Methods.

• Movie 1 -

  Movie 1. LPA stimulates trailing-edge retraction in fibroblasts. NIH3T3 cells stably expressing β−actin−mRFP were plated on fibronectin-coated dishes in the presence of serum as described in Fig. 1. The following day, cells were serum-starved for 12 hours and stimulated with 2 µM LPA. After stimulation, images were recorded for 6 hours at 5-minute intervals using phase-contrast microscopy with ×32 objective power.

• Movie 2 -

  Movie 2. Serum-starved fibroblasts exhibit stable adhesions. NIH3T3 cells stably expressing paxillin-GFP were plated on fibronectin-coated dishes in the presence of serum, as described for Fig. 2. Following the serum starvation paxillin-GFP-rich adhesions were monitored using TIRF microscopy for a period of 7 minutes.

• Movie 3 -

  Movie 3. LPA induces movement of adhesions. NIH3T3 cells stably expressing paxillin-GFP were plated on fibronectin-coated dishes in the presence of serum as described for Fig. 2. The cells were serum-starved for 12 hours and subsequently stimulated with 2 µM LPA. Following LPA stimulation paxillin-GFP-rich adhesions were monitored for a period of 7 minutes using TIRF microscopy. Arrows indicate LPA-induced adhesion movement.