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. Author manuscript; available in PMC: 2008 May 30.
Published in final edited form as: Free Radic Biol Med. 2006 Jul 15;41(8):1315–1324. doi: 10.1016/j.freeradbiomed.2006.07.014

Fig. 1.

Fig. 1

Reduced nonprotein thiols inhibit activation of pro-MMP-9 by a mechanism that entails S-thiolation. (A) The effects of nonprotein thiols on APMA-mediated activation of pro-MMP-9 were determined by gelatin zymography. The enzyme-digested bands are observed as white bands against a dark background. Lane assignments: (1) pro-MMP-9, (2) pro-MMP-9 + 2.5 mM GSH, (3) pro-MMP-9 + 2.5 mM NAC, (4) pro-MMP-9 + 2.5 mM GSSG, (5) pro-MMP-9 + APMA, (6) pro-MMP-9 + 2.5 mM GSH + APMA, (7) proMMP-9 + 2.5 mM NAC + APMA, (8) pro-MMP-9 + 2.5 mM GSSG + APMA. (B) The nature of the reduced thiol pro-MMP-9 interaction was assessed by inclusion of the biotinylated GSH analogue, BioGee, followed by introduction of the disulfide-reducing agent, DTT (blot a). Western blot analyses were then conducted on the same washed membrane to confirm the integrity of the proMMP-9 protein (blot b). Lane assignments: (1) pro-MMP-9 only, (2) pro-MMP-9 + 100 μM BioGee, (3) pro-MMP-9 + 100 μM BioGee + 2 mM DTT, (4) proMMP-9 + 100 μM BioGee + 3 mM DTT, (5) pro-MMP-9 + 100 μM BioGee + 5 mM DTT, (6) pro-MMP-9 + 100 μM BioGee + 10 mM DTT. (C and D) Gelatin zymography was used to assess the effects of reduced thiols on release and subsequent activation of MMP-2 and MMP-9 which was secreted into the conditioned medium by cultured HNSCC cells as well as (E) cells isolated from AIDS-related Kaposi sarcoma (AIDS-KS). To enhance cellular release of MMPs, HNSCC and AIDS-KS cultures were serum deprived (SD) for 48 h, and conditioned medium was collected during this time. Lane assignments: (C) (1) HNSCC 4 48 h SD, (2) HNSCC 4 24 h SD + 24 h 25 mM NAC, (3) HNSCC 4 48 h SD + APMA, (4) HNSCC 4 24 h SD + 24 h 25 mM NAC + APMA, (5) MMP-2/MMP-9 standards; (D) (1) HSCC 9 24 h SD, (2) HNSCC 9 24 h SD + 24 h + 25 mM NAC, (3) HNSCC 9 48 h SD + APMA, (4) HNSCC 9 24 h SD + 24 h 25 mM NAC + APMA, (5) MMP-2/MMP-9 standards; (E) (1) KS 48 h SD, (2) KS 24 h SD + 25 mM NAC, (3) KS 48 h SD + APMA, (4) KS 24 h SD + 24 h 25 mM NAC + APMA, (5) MMP-2/MMP-9 standards.