Fig 2: Relative mRNA transcription of matrix metallopeptidase 2 (MMP2) (A,B) and MMP9 (C,D) in follicular phase (FP) and mid-luteal phase (MLP) mare endometrial explants treated for 24 or 48 h with medium alone (control), elastase (ELA: 0.5 μg/mL), sivelestat (SIV: 10 μg/mL), or ELA (0.5 μg/mL) + SIV (10 μg/mL). Data are shown as median with interquartile range. Results were considered significant at p < 0.05. Different superscript letters indicate significant differences between treatments within each treatment time (a,b—24 h; x,y—48 h). Asterisks indicate statistical differences between times of treatment for the same treatment (* p < 0.05).

Fig 3: HAT‐L4 and MMP‐2 activation. (A) Effects of GM6001 on shNC and shH cells in Matrigel invasion. Results are mean ± SEM and analyzed using Student's t test. (B) Conditioned media from HeLa cells (positive control) and THP‐1–derived shNC, shH and shR cells were analyzed using zymography. Recombinant human MMP‐2 protein (rMMP‐2) was used as another positive control. (C) Western blotting of HAT‐L4 protein in CHO cells transfected with a control vector or HAT‐L4–expressing plasmid. GAPDH expression was used as a control. (D) MMP‐2 activity in the conditioned media from parental CHO (CHO), vector–transfected CHO (CHO/V) and HAT‐L4–expressing CHO (CHO/HL4) cells incubated with (+) or without (−) recombinant pro‐MMP‐2 was analyzed using a fluorogenic assay. Data were analyzed by one‐way ANOVA. (E) qPCR analysis of MMP‐2 mRNA levels in THP‐1 cells transduced with nontargeting shRNAs (shNC) or shRNAs targeting the MMP‐2 gene (shMMP‐2). (F) qPCR analysis of HAT‐L4 mRNA levels in THP‐1 cells transduced with shNC or shMMP‐2. (G) Matrigel invasion of THP‐1 cells transduced with shNC or shMMP‐2. Data in (A), (E), (F) and (G) were analyzed using Student's t test. Data in (D) were analyzed using one‐way ANOVA

Fig 4: Concept of a novel drug design strategy for a MMP-2 activity-dependent anchoring probe (MDAP).

Fig 5: Benchmark of HTPS performance with different proteases.a Peptides generated from benchmark measurements using well-characterized proteases, WN NS3 protease and MMPs; each protease is characterized by the average number of peptides identified from three independent replicate experiments (left) and by the overlap across triplicates (right). For all proteases except WN NS3 (n = 1) three independent replicates were analyzed. Data are presented as mean values±SD. b Specificity benchmark with proteases commonly used in proteomics workflows as well as WN NS3 protease and MMPs presented as iceLogos. The protease specificity preferences are shown for P3-P3’ positions. c Identified cleavages for MMP2 and MMP3 using different protease characterization approaches (PICS43,44, TAILS45, DIPPS21 and HTPS). HTPS analysis was performed with three independent replicates. d MMP2, MMP3 substrate specificity presented as iceLogos covering P3-P3’ positions. e Correlation of the reported specificity enrichment per position for MMP2 and MMP3 between HTPS and other protease workflows (DIPPS21, PICS43,44 and TAILS45). Source data are provided as a Source Data file.