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. 2021 Mar 3;9(5):e14778. doi: 10.14814/phy2.14778

FIGURE 4.

FIGURE 4

Membrane‐bound Mmp‐8 on murine macrophages is resistant to inhibition by Timps but not a low molecular weight synthetic Mmp inhibitor: In A, activated WT peritoneal macrophages were fixed, and then incubated with 500 nM Timp‐1 (n = 7 cell preparations) or 1 mM 1,10‐o‐phenanthroline (1,10‐phen; a low molecular weight non‐selective metalloproteinase inhibitor; n = 3 cell preparations) or no inhibitor (n = 7 preparations, as a control) for 2 h at 37°C. Residual surface Mmp activity was measured using a sensitive, quenched, fluorogenic peptide substrate for Mmps (McaPLGLDpaAR) for 4 h at 37°C and cleavage of this substrate was quantified using fluorimetry. The data are expressed as a % of the no inhibitor control. The boxes in the box‐plots show the medians and 25th and 75th percentiles and the whiskers show the 10th and 90th percentiles. Asterisk indicates p < 0.05 versus the no inhibitor control. In B, equal numbers of bone marrow‐derived macrophages from WT mice were incubated at 4°C with: (1) no exogenous murine Mmp‐8 for 2 h; (2) 200 nM amino‐phenyl mercuric acetate (APMA)‐activated purified murine Mmp‐8 for 2 h; (3) 200 nM APMA‐activated purified murine Mmp‐8 for 1 h followed by 400 nM murine Timp‐1 for 1 h; (4) 200 nM APMA‐activated purified Mmp‐8 for 1 h and then 20 µM GM6001 (a low molecular weight non‐selective metalloproteinase inhibitor; Mr 388 Da). The cells were fixed and washed with buffer three times and then incubated at 37°C with type I collagen conjugated to quenched FITC for 18 h. Mmp‐8‐mediated cleavage of substrate was quantified in cell‐free supernatant samples using fluorimetry, as described in Methods. The data are expressed as a % cleavage of the substrate in cells incubated with active Mmp‐8 without inhibitor (n = 4 separate experiments). The boxes in the box‐plots show the medians and 25th and 75th percentiles and the whiskers show the 10th and 90th percentiles. Data were analyzed using a Kruskal‐Wallis One‐Way ANOVA followed by pair‐wise testing with Mann–Whitney U tests. Asterisk indicates p < 0.05 versus the group indicated