Figure 5
MMP2 activity is up-regulated in BRG1 expressing SK-MEL5 cells and promotes invasion through matrigel. A. Detection of MMP and TIMP protein expression by Western blotting. Tubulin is a loading control. B. MMP activity in control (EV) and SK-MEL5 expressing BRG1 was determined by zymography. Cells were cultured in serum free media for 24 hours and the supernatant collected and normalized to cell number. C. Invasion assays were performed using matrigel coated chambers with 5% FBS as a chemoattractant. Control SK-MEL5 cells (EV) and SK-MEL5 cells expressing BRG1 were seeded at a density of 1.25 × 105 cells per well on top of control or matrigel inserts. Percent invasion through matrigel was calculated relative to migration through the control insert. The data shown is the average of two independent experiments done in triplicate. Expression of BRG1 significantly increased invasion (p < 0.01). D. Vehicle treated (DMSO) or BiPS (10μM) treated BRG1 expressing SK-MEL5 cells were subjected to the invasion assay as described in C. The data shown is representative of two independent experiments performed in duplicate. Treatment with the MMP2/MMP9 inhibitor, BiPS significantly inhibited invasion (p < 0.01). E. SK-MEL5 +BRG1 cells were transfected with Acell SMART Pool siRNAs targeting MMP2 or red non-targeting siRNAs and analyzed by Western blotting 120 hours after transfection. F. Control and MMP2 down-regulated SK-MEL5+BRG1 cells were subjected to the invasion assay as described in C, 120 hours after transfection with siRNAs. Invasion assays were performed in triplicate. Down-regulation of MMP2 significantly compromised invasion (p < 0.01).