MMP activity and the effect of the general MMP inhibitor doxycycline hyclate. MMP-2 and MMP-9 activity in serum-free media of transduced Sum102 (A) and MDA-MB-231 (B) cells, respectively, as determined by gelatin zymography. Transduced cells were seeded in 6-wells trays and grown for three days. Media were replaced with conditioned medium (Sum102) or serum-free AIM-V medium (MDA-MB-231) after 24 hours. α-tubulin was used as loading control. One representative experiment of three is shown. (A lane was removed to make the image smaller in B) The relative invasion of transduced cells untreated (gray bars) or treated with doxycycline hyclate (dark gray bars) was measured using the modified Boyden chamber method. Doxycycline (6 μg/mL) was added to the upper chambers immediately after cell seeding. Results are presented as mean relative invasion + SEM of two to three independent experiments. (C) Sum102 cells (n ≥ 8, *p < .05) and (D) MDA-MB-231 cells (n ≥ 6, *p < .05 and **p < .01) with TFPI downregulated. (E) MDA-MB-231 cells with TFPIβ downregulated (n ≥ 6).