Fig. 2

Doxycyline treatment reduces active MMP-2 and -9 levels in primary stromal endometriotic cells. a and b Dose-dependent decrease of latent and active MMP-2 levels following treatment with doxycycline as detected by zymography (a) and in an activity assay (b) in primary stromal endometriotic cells. In contrast to the immortalized epithelial endometriotic cell line 12Z, MMP-9 was also detectable in the supernatants of stromal endometriotic primary cell cultures. Similarly to MMP-2, treatment with doxycycline led to a dose-dependent decrease of active MMP-9 levels, examined by zymography (c) and in activity assays (d). Note that the activity assay specifically detects one MMP (− 2 or − 9) per assay by specific antibody binding and several washing steps (i.e. washing out of potential interfering inhibitors or activators) in the antibody-coated microwells, whereas zymography is performed by direct loading of the supernatants and therefore reflects the situation “as it is”. This is the reason that both different methods were performed and are represented here. It also explains that the graphs of the different methods are not identical. e Representative picture of a zymography gel loaded with the supernatant of primary cell cultures of endometriotic stromal cells (inverse picture). The bands corresponding to pro-MMP-2 and -9 are clearly visible. Note the second band below corresponding to the activated form of MMP-2 and -9 which is also very lightly visible in the untreated control. Doxycycline treatment led to a dose-dependent reduction of levels of pro-MMP-2 and -9. Representation of the mean values and SD of two independent experiments. f Primary stromal cells were automatically counted after treatment with doxycycline compared to the control. The number of adherent cells with normal microscopical shape was identical up to 10 μg/ml of doxycycline compared to the control, whereas reduced cell numbers were counted when treated with 20 μg/ml and 40 μg/ml, which microscopically correlated with more visible detached dead cells and cell debris