Mmp 7

The tested parameters (MMP-7 and MMP-26) were measured with enzyme-linked immunosorbent assay (ELISA) (MMP-7—R&D systems, Abingdon, UK; MMP-26—EIAab Science, Wuhan, China), according to the manufacturer’s instructions. Plasma concentrations of CA 15-3 were measured by chemiluminescent microparticle immunoassay (CMIA) (Abbott, Chicago, IL, USA). The intra and inter-assay coefficient were checked by the manufacturers of the diagnostic kits to comply with standards.

Primary antibodies used for immunostaining included those for EGFP (Santa Cruz Biotechnology), MMP7 (R & D Systems, Minneapolis, MN, USA), cytokeratin (Leica, Solms, Germany), CD34 (Abcam, Cambridge, MA, USA), GRP78 (Enzo Life Sciences, Lausen, Switzerland), cleaved caspase-3, cleaved caspase-4, cleaved caspase-9, cleaved caspase-12, Cox IV (all from Cell Signaling Technology, Danvers, MA, USA); secondary antibodies included goat anti-mouse HRP (Santa Cruz Biotechnology), goat anti-rabbit HRP (Santa Cruz Biotechnology), donkey anti-goat HRP (Santa Cruz Biotechnology), chicken anti-rat AP (Santa Cruz Biotechnology), chicken anti-rabbit AP (Santa Cruz Biotechnology), chicken anti-mouse AP (Santa Cruz Biotechnology), goat anti-rabbit Alexa 488/594 (Invitrogen), rabbit anti-goat Alexa 594 (Invitrogen), chicken anti-rabbit Alexa 594 (Invitrogen) and chicken anti-mouse Alexa 594 (Invitrogen). Immunostaining was performed using these antibodies. The sections were counter-stained with hematoxylin or nuclear fast red (Vector, Burlingame, CA, USA). For double staining, EGFP, MMP7, cytokeratin, CD34 and PAS staining was performed following TUNEL staining or cleaved caspase-3 staining.

To follow the degradation of the pNGs by DLS, recombinant human MMP-7 (R&D Systems) were activated at 100 µg mL^-1 with 1 mM p-aminophenylmercuric acetate (APMA) in a solution of 50 mM Tris base, 10 mM CaCl₂, 150 mM NaCl, 0.05% (w/v) Brij-35, and pH 7.5 (TCNB) for 1 h at 37 °C. The enzyme solution was diluted to 0.4 µg mL^-1. 50 µL of NGs solution at 1 mg mL^-1 were filled into low volume cuvettes (Sarstedt) and placed in the Zetasizer Nano-ZS 90 (Malvern). The reaction was initiated by addition of enzyme solution (50 µL at 0.4 µg mL^-1). The particle size distributions were measured every 30 min at 37°C over 16 h.

For Co-IP experiment, HEK293T cells were transfected with GFP-tagged MMP7 and Myc-tagged p14^ARF plasmids. Cell lysates were collected 48 hrs post-transfection in RIPA buffer (1XPBS, pH 7.4, 2 mM EDTA, 1% Triton X-100, 0.5% sodium deoxycholate, and protease inhibitor), and used for immunoprecipitation by incubating with anti-Myc or anti-GFP antibodies for 1hr in cold room [26 (link)]. The A/G plus agarose beads were used for immunoprecipitation of antibody-antigen complex, and IgG was used as a control. The reactions were stopped by adding SDS loading buffer, and samples were subject to Western blotting. For IF experiment, cells were grown on coverslips for 2 days and fixed for 15 min in ice-cold methanol. Cells on slides were probed with following antibodies: p14^ARF (14P02, 1:200, NeoMarkers), MMP7 (monoclonal, JL07, sc80825, 1:50, Santa Cruz) or MMP7 (polyclonal, AF907, 1:200, R & D system) overnight at 4°C, followed by incubation with Alexa Fluor 568 or 488 dye conjugated to anti-mouse or rabbit IgG (Invitrogen) antibodies. Slides were washed and mounted with Vectashield medium (Vector Laboratories), and images were scanned with confocal microscopy [38 (link)]. p14^ARF knockdown in PC3 cells were generated using shRNA as described previously [16 (link), 26 (link)].