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. 2015 May 27;6(25):21406–21420. doi: 10.18632/oncotarget.4011

Figure 3. PinX1 inhibits migration and invasion of ccRCC cells by suppressing MMP-2 expression and activity.

Figure 3

a. Western blotting of PinX1, MMP-2, MMP-9, TIMP-1 and TIMP-2 from ccRCC cells transfected with the PinX1 siRNA or control siRNA. MMP-2 expression was up-regulated independent of TIMP-2 in PinX1 knockdown ccRCC cells. b. Western blotting of PinX1, MMP-2, MMP-9, TIMP-1 and TIMP-2 from ccRCC cells transfected with the pEGFP-C3-PinX1 plasmid or vector control. MMP-2 expression was down-regulated independent of TIMP-2 in PinX1 overexpression ccRCC cells. c. top panel, Gelatin zymography analysis of the enzyme activity of MMP-2 in PinX1 knockdown and control group for both 786-O and ACHN cell lines (gels were incubated for 10 h for 786-O cells and 48 h for ACHN cells). Bottom panel, Gelatin zymography analysis of the enzyme activity of MMP-2 in PinX1 overexpression and control group for both 786-O and ACHN cell lines (gels were incubated for 16 h for 786-O cells and 96 h for ACHN cells). The MMP-2 enzyme activity was significantly enhanced after PinX1 overexpressing in ccRCC cells, conspicuously suppressed after PinX1 knockdown. d. Western blotting of PinX1 and MMP-2 from ccRCC cells transfected with the control siRNA, PinX1 siRNA or co-treated with MMP-2 selective inhibitor I (10 μM). The enhancement of MMP-2 expression regulated by PinX1 knockdown in ccRCC cells was blocked by MMP-2 inhibitor I. e. Gelatin zymography analysis of the enzyme activity of MMP-2 in ccRCC cells transfected with the PinX1 siRNA or co-treated with MMP-2 selective inhibitor I (10 μM). The enhancement of MMP-2 activity regulated by PinX1 knockdown in ccRCC cells was blocked by MMP-2 inhibitor. f. and g. The enhancement of migration and invasion regulated by PinX1 knockdown in ccRCC cells was blocked by MMP-2 inhibitor I. All experiments were carried out in triplicate. Histograms represent means ± SD. ***, P < 0.001.