TIMP protein levels determine IR-enhanced MMP activity. A , effectiveness of protein downregulation by siRNA was assessed in the CM (96 h after transfection) and in cell lysate (L) by western blotting; representative blots are shown. A densitometric analysis of the TIMP-levels in the cell lysates, normalized to the loading control, was performed. B, MMP-activity in CM derived from siLuc-, siTIMP-1- and siTIMP-2-transfected HT1080 cells treated with 0.2 nM patupilone and 10 Gy IR, n ≥ 4. Cell treatment started 72 h after siRNA-transfection. C, HT1080 cells were incubated with anti-TIMP-2 or isotype control antibodies in parallel with patupilone (0.2 nM) and IR (10 Gy), n ≥ 4. D, CM, derived from HT1080 cells treated with patupilone (0.2 nM) and IR (10 Gy), was incubated with APMA for 1 h, n = 5. The fold increase in MMP activity was normalized to the activity in patupilone-untreated and sham-irradiated cells (B, C) and APMA-treated cells (D).*P < 0.05, **P < 0.01, ***P < 0.001.