Calpains participate in the expression, activity and secretion of MMP2 and MMP9.

<p>(A) C643 cells were transfected with negative control or with S1P₂ siRNA for 48 h, and then stimulated with 100 nM S1P for 6 h, and the calpain activity was measured. (B) C643 cells were treated with the S1P₂ antagonist JTE-013 (10 μM for 1 h) and then stimulated with S1P (100 nM for 6 h), and the calpain activity was measured. (C) C643 cells were pre-incubated with calpain inhibitor ALLN (50 μM for 1 h) and allowed to invade through collagen IV towards 5% lipid-stripped FBS in the presence or absence of 100 nM S1P for 6 h. The data are presented in the graph. (D) C643 cells were pre-incubated with calpain inhibitor ALLN (50 μM for 1 h) and then stimulated with 100 nM S1P for 6 hours. The expression of MMP2 was then determined. Representative western blots are shown. β-Actin was used as loading control, and the normalized data are presented in respective graphs. (Ea,b) C643 cells were pre-incubated with calpain inhibitor ALLN (50 μM for 1 h) and then stimulated with S1P for 6 h. The medium was collected and the zymography assay was performed. A representative zymography blot is shown. The data were normalized to the protein content on the plates and are presented as graphs. (F) C643 cells were pre-incubated with calpain inhibitor ALLN (50 μM for 1 h) and then stimulated with S1P for 6 hours. The medium was collected and the secreted MMP2 was measured. A representative western blot is shown. The data were normalized to the protein content on the plates. The normalized results in the graphs are the mean ±S.E.M, n = 3–6. Asterisks (*) denote the statistically significant differences compared with respective control. Data were analyzed with one-way ANOVA and Bonferroni’s post hoc test (* <i>P</i> < 0.05; ** <i>P</i> < 0.01; *** <i>P</i> <0.001).