Figure 3
Effects of RA-XII on matrix degradation.
Representative western blots showed the effect of RA-XII on the expressions of ECM-associated proteinases, MMP-2, MMP-9, TIMP-1 and TIMP-2 in 4T1 cells (A) at various time-points with 100 nM of RA-XII and (B) at different doses for 24 hours. The cropped blots have been obtained under the same experimental conditions and the full-length blots were shown in Supplementary information. The histograms showed the quantified results of the expressions of the ECM-associated proteinases, which were normalized with corresponding β-actin protein expression and expressed as fold of untreated controls (mean + SD of 3 independent experiments). (C,D) 4T1 cells were treated with or without RA-XII at 12.5 to 50 nM for 48 hours and conditional medium were subjected to (C) gelatin zymography and (D) uPA activity assay. In gelatin zymography, representative photographs showed the activities of MMP-2 and MMP-9 on the stained gelatin gel. The effects on MMPs activities were represented by the digested gelatin on the gel in uPA chromogenic activity assay, uPA activity was measured using a specific uPA substrate releasing a coloured chromophore. Data were expressed as fold of untreated controls (mean + SD of 3 independent experiments with duplicates each). Statistical differences were determined by One-way ANOVA, followed by Dunnett Test, with *p < 0.05, **p < 0.01, ***p < 0.001 against untreated controls.
