(A) MDA-MB-231 and 4T1 cells were respectively treated with 0, 0.25, and 0.50 µM of casticin for 24 h, and the culture medium was then subjected to gelatin zymography to analyze the activity of MMP-2/9. (B) The activity of MMP-2/9 was separately quantitated as described in the ‘Materials and methods’ section, and normalized to that of the control. (C) The mRNA levels of MMP-2/9 were determined with real-time quantitative RT-PCR after the cells were incubated with 0, 0.25, and 0.50 µM of casticin for 24 h. (D) Western blot analysis of the protein levels of MMP-2/9 in the cells treated with 0, 0.25, and 0.50 µM of casticin for 24 h. β-actin served as an internal control for the protein level. (E) The relative protein levels of MMP-2/9 were quantitated against the densitometric signal of the β-actin bands. Data are expressed as the mean ± S.D. of three independent experiments. *P<0.05 and **P<0.01 compared with the control.