Fig. 1.

Doxycycline reduces active MMP-2 levels in 12Z endometriotic cells. a Determination of the active form of MMP-2 in doxycycline-treated 12Z endometriosis cells in an activity assay (Biotrak MMP-2 activity assay) which specifically binds MMP-2 in antibody-coated microwells followed by repeated washing steps. The treatment with doxycycline reduced the active MMP-2 levels in a dose-dependent manner. The mean values of three independent treatments are represented with the standard error of mean (SEM) and significance labeled as “α” if p ≤ 0.05. b Cell viability was assessed with trypan blue to exclude that reduced MMP-2 levels were due to increased cell death rates. As is shown in the figure, concentrations of doxycycline up to 20 μg/ml did not lead to reduced cell viability. Representation of the mean and SEM, significant differences (i.e. if p ≤ 0.05) are labeled with an “α” next to the bar. c Representative illustration of a zymography gel (inverse picture) for detection of latent and active gelatinase levels ((pro-)MMP-2 and -9). The analysis was performed in culture supernatants of immortalized 12Z endometriotic cells treated with respective doses of doxycycline in serum-free medium for 24 h. Samples were loaded on an electrophoresis gel containing gelatin, followed by an incubation for 24 h in a buffer medium containing zinc and calcium chloride at 37 °C. The molecular size of the detected bands corresponds to pro MMP2 and the bands showed decreasing expression with increasing doses of doxycycline. Note the second band below pro MMP2 corresponding to active MMP-2 which is not visible in the samples probably due to the very low amount compared to the latent form. (Pro-)MMP-9 was not detectable in 12Z by gelatin zymography. rMMP-2 and -9: reconstituted lyophilized human pro-MMP-2 and pro-MMP-9. d Quantification of pro-MMP-2 activity from two independent drug treatments. Representation of the mean and standard deviation (SD)