Skip to main content
. 2012 Sep 5;7(9):e44618. doi: 10.1371/journal.pone.0044618

Figure 4. Knock-down of SPARC expression in HGC-27 cells promotes angiogenesis via up-regulated VEGF and MMP-7 expression.

Figure 4

(A) Conditioned media from HGC-P, HGC-EV, HGC-sh with or without rhSPARC (0.3 µg/ml) and HGC-sh+MMP7-sh cells were concentrated under the same conditions. β-casein zymography was conducted for MMP-7 activity. Western blotting analysis was performed for MMP-7, SPARC and VEGF protein levels in conditioned media. The cells were collected and lysates probed with GAPDH antibody to calibrate total amount of the respective proteins. Columns are means (±s.d.) of triplicate experiments. (B) In vitro angiogenesis: To confirm that SPARC expression-mediated anti-angiogenic effects are due to altered MMP-7 and VEGF expression rather than to the expression of SPARC itself, harvested supernatant from HGC-sh cells was added to 0.3 µg/ml rhSPARC. Supernatants from both of HGC-sh and HGC-sh+MMP7-sh cells with neutralising antibody to VEGF were also used in co-culture assay (anti-VEGF =  neutralising antibody to VEGF). HUVECs were seeded in Matrigel-coated 96-well plates incubated with conditioned media. The effects of conditioned media on the pre-formed tubes of HUVECs were analysed, and the tube length was measured. Tube length data shown are the means (±s.d.) of quadruplicate determinations from three separate experiments. *P<0.05, significant difference from HGC-P cells, **P<0.05, significant difference from HGC-sh cells.