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. Author manuscript; available in PMC: 2014 Sep 19.
Published in final edited form as: J Invest Dermatol. 2012 Feb 16;132(5):1435–1442. doi: 10.1038/jid.2012.14

Figure 4. Doxycycline inhibits cellular and secreted trypsin-like serine protease (TLSP) activity of live keratinocytes.

Figure 4

(a) To examine the activity of doxycycline on TLSP activity produced by live normal human epidermal keratinocytes (NHEKs), cells were cultured in media containing doxycycline (225 µm) for 18 hours. The matrix metalloproteinase (MMP) inhibitors SB3CT (5 µm) and GM6001 (25 µm) were used as positive controls for MMP inhibition. TLSP activity in cell lysate following culture with doxycycline was determined with TLSP-specific substrate (relative fluorescence units (RFU)). Doxycycline significantly attenuated cellular TLSP activity generated by live cells. Incubating cells with SB-3CT and GM6001 also significantly decreased cellular TLSP activity. (b) Secreted TLSP activity was similarly assayed in NHEK conditioned media. Doxycycline significantly decreased secreted TLSP activity. The inability of doxycycline to inhibit TLSP activity when directly added to enzyme extracts (Figure 3c) suggests that the inhibition induced in live cells is by an indirect mechanism and is related to the capacity to inhibit MMPs. *P<0.05, **P<0.01 by Student’s t-test, versus vehicle or control.