Effect of PCs inhibition on gelatinase activity and MMP-2/TIMP-1 and MMP-2/TIMP-2 ratios.

<p>(<b>A</b>) Serum-free media derived from M10 and M10/PDX cells were collected and analyzed for gelatinase enzymatic activity as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009992#s2" target="_blank">Materials and Methods</a>. Following total RNA extraction, real-time PCR analysis was performed using specific primers for MMP-2 (<b>B</b>), TIMP-1 (<b>C</b>), TIMP-2 (<b>D</b>) or β2-microglobulin as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009992#s2" target="_blank">Materials and Methods</a>. During PCR, the transcription of β2-microglobulin that was evaluated in each sample was used as endogenous control. Results are expressed as mRNA transcript ratios. Data are shown as means ± S.E of three experiments performed in duplicate. (<b>E, F</b>) The ratios of MMP-2/TIMP-1 and MMP-2/TIMP-2 are deduced from data obtained in (B), (C) and (D). (<b>G, H</b>), Tumor cells were incubated at 37°C in serum-free media. After 24 hours, media were collected and analyzed for the presence of TIMP-1 and TIMP-2 using ELISA kit. Results shown are representative of 3 experiments. Data are mean ± SEM (<i>n = </i>6 per group). Student's <i>t</i> test was used for statistical analysis ***, <i>P</i><0.001.