Fig. 2.

Calpains are activated by growth factors and result in vimentin cleavage. a Invading cultures were treated with indicated doses of CI and allowed to invade collagen matrices for 22 h in the presence of S1P and GF. Whole cell extracts were subjected to Western blotting and probed with antisera directed to vimentin. Blots were stripped and re-probed with GAPDH- specific antisera. Results are representative of three independent experiments. b ECs were plated at 80% confluence in a 96 well plate in M199 medium containing RSII for 8 h. The cells were pre-treated with 31.6 μM CI or DMSO (CON) for 1 h and then loaded with 30 μM of the calpain substrate tBoc-LM-CMAC. Cells were treated without (CON) or with S1P (1 μM), GF (40 ng/ml VEGF and bFGF) or S1P + GF for 30 min and imaged. Calpain activity was quantified as indicated in the “Materials and methods” section. Results are representative of four independent experiments. Data shown are average values ± SD. *P < 0.05, and **P < 0.01 compared to Control, ***P < 0.001 compared to S1P + GF by Student’s t test. c Endothelial cells were seeded on 3D collagen matrices and allowed to invade for 4 or 6 h. Whole cell lysates were prepared and analyzed by Western blotting with antisera directed to vimentin and GAPDH control. d Quantification of intensities of vimentin cleavage products with treatment conditions. Data are derived by averaging band intensities from three independent experiments. **P < 0.01 versus CON; ^‡ P < 0.05 versus all other treatments by Student’s t test