Figure 1.

TIMP-2 expression levels, MMP-2 and TIMP-2 activities in A549 cell lines, A549 WT (wild-type, parental), EV (empty vector control), T2 (TIMP-2) and Ala+T2 (Ala+TIMP-2 mutant lacking MMP-2 inhibitory activity). A: Total RNA was extracted from the retrovirally transfected A549 cells and used to determine TIMP-2 mRNA levels using quantitative real-time RT-PCR. Fold expression mRNA levels are relative to those of the house-keeping gene GAPDH. Error bars represent the SEM from seven independent experiments. B: Supernatant from cultured A549 stable cells was collected 48 hours after culture and used to determine secreted TIMP-2 protein levels using a human TIMP-2 enzyme-linked immunosorbent assay (ELISA). TIMP-2 concentrations (ng/mL) were adjusted to the number of cultured cells. Error bars represent the SEM from five independent experiments. C: Conditioned media (CM), prepared as described above, were analyzed in gelatin zymography to determine MMP-2 (gelatinase A) activity in the A549 stables. MMP-2 shows two bands, the 72-kDa latent zymogen (pro-MMP-2) and the 66 kDa active enzyme (active MMP-2). A 4 ng quantity of recombinant gelatinase A (rGel-A) was run as a positive control. D: CM were analyzed in reverse zymography to determine TIMP-2 inhibitory activity in the A549 stables. Both TIMP-1 (28.5 kDa) and TIMP-2 (21.7 kDa) are present in the CM. Recombinant proteins (rTIMP-1 and rTIMP-2) were run as controls at the indicated amounts. For Figures 1C and 1D: Relative band intensities are shown below each figure as compared with A549 WT. Each zymogram is representative of at least three independent experiments. M = Molecular Weight Marker.