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. Author manuscript; available in PMC: 2008 Jul 12.
Published in final edited form as: Exp Biol Med (Maywood). 2008 Apr 29;233(7):860–873. doi: 10.3181/0801-RM-12

Figure 3.

Figure 3

Characterization of MMP9 and SPARC expression in PAN02-PRV and PAN02-MMP9 cells. A) Gelatinolytic activity in total cell extracts of PAN02-PRV (PRV) and PAN02-MMP9 (MMP). Three serial dilutions of RIPA extracts corresponding to 5, 1, and 0.2 μg of protein were loaded on an 8% gelatin-containing SDS gels. B) Gelatinolytic activity in conditioned medium from the same cells as in (A) after 24 h of serum depletion. In panel A and B HT1080 conditioned media (+) was loaded as a positive control of gelatinolytic activity and shows MMP9 and MMP2 bands. C) Membrane and cytosolic fractions were obtained from total cell lysates. 25 μg of total extracts (E) and cytosolic (C) fraction and 1/6 of the membrane fraction (M) were analyzed on a 10% SDS gel. Western blot analysis of MMP9 and SPARC was performed; anti-TfR (membrane marker) and anti-tubulin (cytosolic marker) were used as controls for fractionation. D) Confocal microscopy of MMP9 (red) in PAN02 cells, cells transduced with the indicated construct are positive for GFP (green). E) PAN02-PRV (PRV) and PAN02-MMP9 (MMP) cells plated on either collagen I or collagen IV slides were analyzed for SPARC (red) expression by immunocytochemistry. Cells transduced with the indicated construct are positive for GFP (green). F) PAN02-PRV (PRV) and PAN02-MMP9 (MMP) cells were analyzed for gelatinase activity (green) by incubating with DQ-gelatin and subsequent confocal microscopy. To-Pro3 cell tracker (red) was used to localize nuclei. G) Quantification of gelatinase activity in PAN02-PRV (PRV) and PAN02-MMP9 (MMP9). Cells were incubated with DQ-gelatin for the indicated time and the fluorescence increment was represented. Values are normalized to PAN02-PRV cells after 3h of incubation (** p<0.01, Students t test).