JCS036152 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Average integrated traction forces of MEF cells following 10 minute treatment with 15 µM Calpeptin, 65 µM MDL or DMSO. Bars represent data from 3-4 cells each, collected every 5 minutes for 1 hour.

• Supplemental Figure S2 -

  Fig. S2. Calpain activity assayed using the Biovision assay kit. Relative fluorescence units obtained using the ac-LLY-AFC substrate and 50 µg protein lysate from MEF cells deficient in capn4 or overexpressing calpastatin. The true extent of Calpain activity is likely to be less as the assay kit does not differentiate between members of the Calpain family nor does it fully eliminate the activities of other proteases that might recognize the provided substrate.

• Supplemental Figure S3 -

  Fig. S3. Actin and vinculin immunofluorescence of fibroblasts after siRNA-mediated silencing of capn1 (A,B) and capn2 (C,D) and calpastatin overexpression (E,F) show typical stress fiber and focal adhesions organization and morphology. Scale bar: 10 µm.

• Movie 1 -

  Movie 1. Response of rescued capn4^−/− cells to the stimulation of pushing forces, exerted with a blunted microneedle on the flexible substrate. Duration, 45 minutes.

• Movie 2 -

  Movie 2. Failure of capn4^−/− cells to respond to the stimulation of pushing forces, exerted with a blunted microneedle on the flexible substrate. Duration, 40 minutes.

• Movie 3 -

  Movie 3. Response of control fibroblasts transfected with scrambled small RNA to pushing forces, exerted with a blunted microneedle on the flexible substrate. Duration, 35 minutes.

• Movie 4 -

  Movie 4. Failure of fibroblasts to respond to the stimulation of pushing forces exerted with a blunted microneedle on the flexible substrate, after capn1 has been silenced by siRNA. Duration, 45 minutes.

• Movie 5 -

  Movie 5. Fibroblasts whose capn2 gene has been silenced by siRNA fail to respond to mechanical stimulation. Duration, 50 minutes.