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. 2014 Nov 21;29(1):53–67. doi: 10.1210/me.2014-1201

Figure 5. Effect of SphK, SMase, and S1P lyase inhibition and extracellular S1P addition on RLX-induced MMP-9 and MMP-2 activity in primary cardiac muscle cells and H9c2 cells.

Figure 5.

A and B, Representative zymographs of latent (L)-MMP-9, active (A)-MMP-9, and MMP-2 activity from conditioned media obtained from primary cardiac muscle cells (A) and H9c2 cardiomyoblasts (B) treated for 30 minutes with S1P (1μM), compound II (iSK, 5μM), GW9846 (10μM), or THI (6.5μM) and then incubated in the absence or presence of RLX (50 ng/mL) over 24 hours. Relative OD was determined by densitometry scanning and data as relative percentage to control (vehicle) arbitrarily normalized to 100 are reported in the graphics. Data are mean ± SEM of at least 3 independent experiments (Student's t test; *, P < .05 vs untreated cells (vehicle); §, P < .05 vs RLX). C and D, MMP-9 ELISA. Conditioned cell media obtained from primary cardiac muscle cells (C) and H9c2 cells (D) treated for 30 minutes with S1P (1μM), compound II (iSK, 5μM), GW9846 (10μM), or THI (6.5μM) and then incubated in absence or presence of RLX (50 ng/mL) over 24 hours. Samples were analyzed and data reported as nanograms per milliliter as described in Materials and Methods. Data are mean ± SEM of at least 3 independent experiments (Student's t test; *, P < .05 vs untreated cells (vehicle); §, P < .05 vs RLX).